The immunology of HIV-infected long-term non-progressors--a current view.

Long-term non-progressors (LTNP) are human immunodeficiency virus (HIV)-infected individuals characterized by the absence of disease, low viral loads and stable or even increasing CD4(+) T cell counts for prolonged periods of time. In these subjects, an HIV-specific immune response which is either stronger or directed against a wider array of viral epitopes than that seen in progressors, can be often detected. Here, we summarize the characteristics of HIV-specific CD4(+) and CD8(+) T cell responses in LTNP, and discuss how a highly effective T cell-mediated immune response against HIV might contribute to the establishment of this particular condition.

Apoptotic cells overexpress vinculin and induce vinculin-specific cytotoxic T-cell cross-priming.

Here we show that apoptotic cells overexpress vinculin and are ingested by dendritic cells, which subsequently cross-prime vinculin-specific cytotoxic T lymphocytes (CTLs). Successful cross-priming requires that the apoptotic cells provide maturation signals to dendritic cells through CD40-CD40 ligand (CD40L) interactions. If apoptotic cells are CD40L-, the help of a third T cell is needed for priming, indicating a regulatory role for apoptotic cells in determining priming or tolerance. Vinculin-specific CTL priming is also related to apoptosis in vivo, given that in HIV-seropositive individuals, the frequency of specific CTLs depends on the proportion of peripheral CD40L+ apoptotic cells.

Spreading of HIV-specific CD8+ T-cell repertoire in long-term nonprogressors and its role in the control of viral load and disease activity.

Long-term non-progressors (LTNP) represent a minority of human immunodeficiency virus (HIV) infected individuals characterized by stable or even increasing CD4+ T-cell count and by stronger immune responses against HIV than progressors. In this study, HIV-specific effector CD8+ T cells, as detected by both a sensitive ex vivo enzyme-linked immunospot (ELISPOT) assay and specific major histocompatibility complex (MHC) peptide tetramers, were at a low frequency in the peripheral blood of LTNP, and recognized a lower number of HIV peptides than their memory resting cell counterparts. Both factors may account for the lack of complete HIV clearance by LTNP, who could control the viral spread, and displayed a higher magnitude of cytotoxic T lymphocyte (CTL) responses than progressors. By combining cell purification and ELISPOT assays this study demonstrates that both effector and memory resting cells were confined to a CD8+ population with memory CD45RO+ phenotype, with the former being CD28- and the latter CD28+. Longitudinal studies highlighted a relatively stable HIV-specific effector repertoire, viremia, and CD4+ T-cell counts, which were all correlated with maintenance of nonprogressor status. In conclusion, the analysis of HIV-specific cellular responses in these individuals may help define clear correlates of protective immunity in HIV infection.

Virus-specific CD8(+) T cells with type 1 or type 2 cytokine profile are related to different disease activity in chronic hepatitis C virus infection.

The present study demonstrates that the quality of the virus-specific CD8(+) T cell responses, as detected by both enzyme-linked immunospot assay and specific MHC-peptide tetramers, changed in relation to the different disease activity in chronically hepatitis C virus-infected patients. Indeed, both the serum alanine transaminase and the hepatic flogosis levels were related directly to the frequencies of peripheral memory effector CD8(+) T cells producing IFN-gamma (Tc1), but inversely to the frequencies of those producing both IL-4 and IL-10 (Tc2). Longitudinal studies highlighted that Tc1 or Tc2 responses fluctuate in relation to the different phases of the disease in the same individual. Furthermore, the Tc1 or Tc2 phenotype correlates with tetramer-positive cells expressing either CXCR3 or CCR3, promoting differential tissue localization of these cells and the maintenance of T cell homeostasis. Finally, studies at the level of liver-infiltrating lymphocytes indicated that they produced both IFN-gamma and IL-4 with an evident bias towards the Tc1-like phenotype. Our studies suggest that the progressive fluctuation of Tc1 and Tc2 responses may play a fundamental role in maintaining a long-lasting low-level liver inflammation, and may constitute the basis for new therapeutic strategies of immune regulation.

Persisting viruses and autoimmunity.

Viral infections can be responsible for the onset and sustaining of autoimmune processes. We discuss how chronic inflammation associated with viral persistence is the prerequisite for initiation of a multi-step process leading to autoimmunity. Firstly, chronic inflammation may favor the priming of autoreactive T cells that have escaped thymic selection and are specific for self-mimicking viral peptides in the periphery. In addition, viral persistence and inflammation can act synergistically to induce and sustain autoimmunity either unveiling cryptic self-epitopes, or favoring determinant spreading, or activating dendritic cells, or promoting constant priming of new autoreactive T cells, or contributing to the efficient generation of effector cells, or, finally, restimulating memory T lymphocytes.

Generation of heterogeneous rabbit anti-DNP antibodies by gene conversion and hypermutation of rearranged VL and VH genes during clonal expansion of B cells in splenic germinal centers.

The mechanisms described here account for development of the heterogeneous high-affinity anti-DNP antibodies that rabbits can produce. Rearranged immunoglobulin light and heavy chain genes from single DNP-specific splenic germinal center B cells were amplified by PCR. We found that in clonal lineages, rearranged V[kappa] and V[H] are further diversified by gene conversion and somatic hypermutation. The positive and negative selection of amino acids in complementarity-determining regions observed allows emergence of a variety of different combining site structures. A by-product of the germinal center reaction may be cells with sequences altered by gene conversion that no longer react with the immunizing antigen but are a source of new repertoire. The splenic germinal center would thus play an additional role in adults similar to that of the appendix and other gut-associated lymphoid tissues of young rabbits.

Gene-conversion in rabbit B-cell ontogeny and during immune responses in splenic germinal centers.

Combinatorial diversity is limited in rabbits because only a few V(H) genes rearrange. Most diversification of the primary repertoire is generated by somatic hypermutation and gene conversion-like changes of rearranged V(H) in B cells that migrate to appendix and other gut associated lymphoid tissues (GALT) of young rabbits. The changes are referred to as gene conversion-like because the non-reciprocal nature of the alterations introduced has not yet been demonstrated. There are many similarities between rabbits and chickens in how their B cells develop and diversify their repertoires. However, although the majority of rabbit B cells may have rearranged and diversified their V genes early in life, some B cells in adult rabbits have rearranged VH sequences that are identical or nearly identical to germline sequences. We found these cells in splenic germinal centers (GC) on days 7 and 10 after immunization of normal adult rabbits with DNP-BGG. By day 15, all rearranged V(H) sequences were diversified. We find an overall pattern of splenic precursor cells whose germline or near germline sequences change both by gene conversion and point mutations during early divisions and mainly by point mutations during later divisions. These events, in parallel with diversification of light chain sequences, may produce the diverse combining sites that serve as substrates for further affinity maturation by selection either within GC or later among emigrant cells in sites such as bone marrow. Some of the sequences altered by gene conversion in splenic germinal centers may also produce new members of the B-cell repertoire in adult rabbits comparable to those produced in GALT of neonatal rabbits.

Gene conversion and hypermutation during diversification of VH sequences in developing splenic germinal centers of immunized rabbits.

The young rabbit appendix and the chicken bursa of Fabricius are primary lymphoid organs where the B cell Ab repertoire develops in germinal centers (GCs) mainly by a gene conversion-like process. In human and mouse, V-gene diversification by somatic hypermutation in GCs of secondary lymphoid organs leads to affinity maturation. We asked whether gene conversion, somatic hypermutation, or both occur in rabbit splenic GCs during responses to the hapten DNP. We determined DNA sequences of rearranged heavy and light chain V region gene segments in single cells from developing DNP-specific GCs after immunization with DNP-bovine gamma-globulin and conclude that the changes at the DNA level that may lead to affinity maturation occur by both gene conversion and hypermutation. Selection was suggested by finding some recurrent amino acid replacements that may contribute increased affinity for antigen in the complementarity-determining region sequences of independently evolved clones, and a narrower range of complementarity-determining region 3 lengths at day 15. Some of the alterations of sequence may also lead to new members of the B cell repertoire in adult rabbits comparable with those produced in gut associated lymphoid tissues of young rabbits.

Analyses of single B cells by polymerase chain reaction reveal rearranged VH with germline sequences in spleens of immunized adult rabbits: implications for B cell repertoire maintenance and renewal.

We used PCR to amplify rearranged VHDJH genes in single cells collected by micromanipulation from splenic germinal centers of immunized adult rabbits. In the course of the study, the objective of which was to analyze diversification of rearranged VHDJH sequences, we were surprised to find cells 7 and 10 days after immunization with rearranged VH1a2 as well as a-negative (y33 and x32) sequences that were identical or close to germline (10 or fewer changes). About 58% (82/140) of the sequences had unique CDR3 regions and were unrelated. In seven different germinal centers, we found one to four different clones with two to seven members. Clonally related cells underwent diversification by hypermutation and gene conversion. We found that contrary to published reports, adult rabbits indeed have newly diversifying B cell receptors in splenic germinal centers. The attractive idea that the rabbit, like the chicken, develops its B cell repertoire early in life and depends upon self-renewing cells in the periphery to maintain its B lymphocyte pool throughout life, is challenged by the current finding. Although a major population of B lymphocytes may be generated early in life, diversified extensively, and maintained by self-renewal in the periphery, some sources of cells with sequences close to germline do exist in adult rabbits and appear in the developing germinal centers. Although considerable repertoire diversity is generated in young rabbits, mechanisms for continued generation of B cell receptor diversity are retained in adult life, where they may confer survival advantage.

High RAD51 mRNA levels in young rabbit appendix. A role in B-cell gene conversion?

The rabbit has a limited number of VH genes that rearrange. As in the chicken, the 3'-most VH1 gene is rearranged in most B lymphocytes. This laboratory reported that by 6 weeks after birth, diversification of rearranged VH genes occurs, at least in part, by gene conversion-like events in the appendix, suggesting that this organ is a homologue of the avian bursa of Fabricius. Rad51 contributes to the repair of double-strand breaks in DNA during somatic and meiotic recombination. The gene was first identified in lower eukaryotes, and later in vertebrates including chicken, as encoding an Escherichia coli RecA-like protein. We report the cloning and sequencing of RAD51 from the rabbit. Because the chicken bursa was shown to express high levels of RAD51 message, we investigated the expression of RAD51 in the rabbit appendix and other tissues. Using a quantitative polymerase chain reaction mimic assay and conventional northern analyses, we found high RAD51 expression in young rabbit appendix comparable to levels in testis where there is an abundance of meiotic recombination. RAD51 levels were three times higher in appendix B lymphocytes compared with T lymphocytes and were lower in adult appendix, as well as in spleen and Peyer's patches of young rabbits. We measured the levels of message in several appendix cell sub-populations obtained by fluorescence-activated cell sorting and found that sub-populations of B lymphocytes corresponding to different stages of B-cell development as well as B cells undergoing isotype switch did not have significantly different mRNA levels.

VH mutant rabbits lacking the VH1a2 gene develop a2+ B cells in the appendix by gene conversion-like alteration of a rearranged VH4 gene.

We investigated the molecular basis for the appearance of V(H)a2 allotype-bearing B cells in mutant Alicia rabbits. The mutation arose in an a2 rabbit; mutants exhibit altered expression of V(H) genes because of a small deletion encompassing V(H)1a2, the 3'-most gene in the V(H) locus. The V(H)1 gene is the major source of V(H)a allotype because this gene is preferentially rearranged in normal rabbits. In young homozygous ali/ali animals, the levels of a2 molecules found in the serum increase with age. In adult ali/ali rabbits, 20 to 50% of serum Igs and B cells bear a2 allotypic determinants. Previous studies suggested that positive selection results in expansion of a2 allotype-bearing B cells in the appendix of young mutant ali/ali rabbits. We separated appendix cells from a 6-wk-old Alicia rabbit by FACS based on the expression of surface IgM and a2 allotype. The VDJ portion of the expressed Ig mRNA was amplified from the IgM+ a2+ and IgM+ a2- populations by reverse transcriptase-PCR. The cDNAs from both populations were cloned and sequenced. Analysis of these sequences suggested that, in a2+ B cells, the first D proximal functional gene in Alicia rabbits, V(H)4a2, rearranged and was altered further by a gene conversion-like mechanism. Upstream V(H) genes were identified as potential gene sequence donors; V(H)9 was found to be the most frequently used gene donor. Among the a2- B cells, y33 was the most frequently rearranged gene.

Endogenous retroviral gp70 genes of the murine lymphoma L5178Y: analysis of restriction fragment polymorphism upon induction of drug-mediated immunogenicity.

Highly immunogenic ("xenogenized") tumor variants appear after treatment of murine lymphoma L5178Y with the triazene derivative DTIC, this phenomenon being associated with the appearance of structurally abnormal gp70 env proteins in the cell variants. In the present study, we have isolated and sequenced several PCR-amplified gp70 cDNA genes from L5178Y cells. One of the resulting clones was used as a probe in Southern and Northern analysis of parental and xenogenized cells. The results indicated that xenogenization of tumor cells is associated with changes in retroviral env sequences detectable at the genomic level.

Mucosal and systemic T helper cell function after intragastric colonization of adult mice with Candida albicans.

In 85% of adult DBA/2 mice inoculated intragastrically with Candida albicans, significant numbers of yeast cells were recovered from the gastrointestinal tract for up to 4 weeks, with the animals eventually clearing infection in the absence of systemic disease despite the occurrence of localized, self-limiting foci of mucosal involvement in their stomachs. Two major findings in colonized mice were defective production of IgA, interleukin (IL)-4, and IL-5 by Peyer's patches lymphocytes and increased numbers of interferon-gamma-producing T cells in mesenteric lymph nodes and spleens. Relatively low levels of circulating antibodies of T helper type 2 (Th2)-dependent isotypes were also found in colonized mice, which exhibited strong footpad responses and increased resistance to systemic reinfection. Unlike systemic challenge, gastrointestinal colonization of adult immunocompetent DBA/2 mice with C. albicans appears to be an effective stimulus for the systemic development of protective Th1 responses.

Natural killer cells do not play a dominant role in CD4+ subset differentiation in Candida albicans-infected mice.

The effects of in vivo administration of monoclonal antibodies against NK-1.1-bearing cells on the early production of gamma interferon (IFN-gamma) in vitro and development of Th1-associated immunity were studied in mice infected with a live vaccine strain of Candida albicans. At 1 and 4 days postinfection, natural killer (NK) cell-enriched fractions from the spleens of antibody-treated mice displayed a dramatic reduction in 5E6+ lymphocytes and negligible anti-YAC-1 cytotoxic activity in vitro. Nevertheless, the frequency of IFN-gamma-producing cells in those fractions was reduced by less than half, on average, by anti-NK-1.1 treatment in vivo. In addition, the antibody-treated and infected mice demonstrated unchanged T helper cell responses, as measured by yeast-specific footpad reactions, resistance to reinfection, occurrence of antibodies of different isotypes, and production in vitro of interleukin-2 (IL-2), IFN-gamma, IL-4, and IL-10 by CD4+ cells. Therefore, although NK cells may contribute to early IFN-gamma production in Candida-vaccinated mice, these cells apparently do not play a dominant role in the qualitative development of yeast-specific T helper responses.

Interleukin-4 and interleukin-10 inhibit nitric oxide-dependent macrophage killing of Candida albicans.

Mouse peritoneal and splenic macrophages treated with interferon-gamma (IFN-gamma) and infected with the yeast Candida albicans expressed high fungicidal activity in vitro that correlated with increased nitrite concentrations in culture supernatants. Both effects were reduced by an inhibitor of nitric oxide (NO) synthesis which, in vivo, impaired the animals' ability to mount a footpad reaction and clear the fungus from infected organs. Because T helper type-2 (Th2) cytokines in candidiasis are known to limit the expression of protective Th1 functions, we tested the effect of interleukin (IL)-4 and IL-10 on candidacidal activity and NO production of IFN-gamma-activated macrophages. Fungal killing and NO secretion were inhibited, in a dose-dependent manner, by the two cytokines either separately or in combination. Impaired candidacidal activity was also demonstrable in the presence of monoiodoacetic acid, an inhibitor of phagocytosis. These data demonstrate that NO is involved in macrophage killing of C. albicans and support the notion that regulation of Th1 effector function by IL-4 and IL-10 might involve modulation of NO synthesis.