Telomere lengthening and telomerase activation during human B cell differentiation

The function of the immune system is highly dependent on cellular differentiation and clonal expansion of antigen-specific lymphocytes. However, little is known about mechanisms that may have evolved to protect replicative potential in actively dividing lymphocytes during immune differentiation and response. Here we report an analysis of telomere length and telomerase expression, factors implicated in the regulation of cellular replicative lifespan, in human B cell subsets. In contrast to previous observations, in which telomere shortening and concomitant loss of replicative potential occur in the process of somatic cell differentiation and cell division, it was found that germinal center (GC) B cells, a compartment characterized by extensive clonal expansion and selection, had significantly longer telomeric restriction fragments than those of precursor naive B cells. Furthermore, it was found that telomerase, a telomere-synthesizing enzyme, is expressed at high levels in GC B cells (at least 128-fold higher than those of naive and memory B cells), correlating with the long telomeres in this subset of B cells. Finally, both naive and memory B cells were capable of up-regulating telomerase activity in vitro in response to activation signals through the B cell antigen receptor in the presence of CD40 engagement and/or interleukin 4. These observations suggest that a novel process of telomere lengthening, possibly mediated by telomerase, functions in actively dividing GC B lymphocytes and may play a critical role in humoral immune response by maintaining the replicative potential of GC and descendant memory B cells.

Identification of proacrosin binding protein sp32 precursor as a human cancer/testis antigen

Serological expression cloning of antigens eliciting a humoral immune response to a syngeneic mouse sarcoma identified pem (mouse placenta and embryonic expression gene) as a new member of the cancer/testis family. To identify the human homologue of pem, mouse pem sequences and pem-related expressed sequence tags from human testis were used as PCR primers for amplification using human testis cDNA. However, rather than pem, another gene, designated OY-TES-1, was isolated and found to be the human homologue of proacrosin binding protein sp32 precursor originally identified in mouse, guinea pig, and pig. OY-TES-1 maps to chromosome 12p12-p13 and contains 10 exons. Southern blot analysis suggests the presence of two OY-TES-1-related genes in the human genome. In normal tissues, OY-TES-1 mRNA was expressed only in testis, whereas in malignant tissues, a variable proportion of a wide array of cancers, including bladder, breast, lung, liver, and colon cancers, expressed OY-TES-1. Serological survey of 362 cancer patients with a range of different cancers showed antibody to OY-TES-1 in 25 patients. No OY-TES-1 sera reactivity was found in 20 normal individuals. These findings indicate that OY-TES-1 is an additional member of the cancer/testis family of antigens and that OY-TES-1 is immunogenic in humans.

Different response to Plasmodium falciparum malaria in West African sympatric ethnic groups

The comparison of malaria indicators among populations that have different genetic backgrounds and are uniformly exposed to the same parasite strains is one approach to the study of human heterogeneities in the response to the infection. We report the results of comparative surveys on three sympatric West African ethnic groups, Fulani, Mossi, and Rimaibé, living in the same conditions of hyperendemic transmission in a Sudan savanna area northeast of Ouagadougou, Burkina Faso. The Mossi and Rimaibé are Sudanese negroid populations with a long tradition of sedentary farming, while the Fulani are nomadic pastoralists, partly settled and characterized by non-negroid features of possible caucasoid origin. Parasitological, clinical, and immunological investigations showed consistent interethnic differences in Plasmodium falciparum infection rates, malaria morbidity, and prevalence and levels of antibodies to various P. falciparum antigens. The data point to a remarkably similar response to malaria in the Mossi and Rimaibé, while the Fulani are clearly less parasitized, less affected by the disease, and more responsive to all antigens tested. No difference in the use of malaria protective measures was demonstrated that could account for these findings, and sociocultural or environmental factors do not seem to be involved. Known genetic factors of resistance to malaria did not show higher frequencies in the Fulani. The differences in the immune response were not explained by the entomological observations, which indicated substantially uniform exposure to infective bites. The available data support the existence of unknown genetic factors, possibly related to humoral immune responses, determining interethnic differences in the susceptibility to malaria.

The CD19 signal transduction molecule is a response regulator of B-lymphocyte differentiation.

The phenotypes of CD19-deficient (CD19-/-) mice, and human CD19-transgenic (hCD19TG) mice that overexpress CD19 indicate that CD19 is a response regulator of B-lymphocyte surface receptor signaling. To further characterize the function of CD19 during B-cell differentiation, humoral immune responses to a T-cell-independent type 1 [trinitrophenyl-lipopolysaccharide (TNP-LPS)], a T-cell-independent type 2 [dinitrophenyl (DNP)-Ficoll], and a T-cell-dependent [DNP-keyhole limpet hemocyanin (KLH)] antigen were assessed in CD19-/- and hCD19TG mice. B cells from CD19-/- mice differentiated and underwent immunoglobulin isotype switching in vitro in response to mitogens and cytokines. In vivo, CD19-/- mice generated humoral responses to TNP-LPS and DNP-KLH that were dramatically lower than those of wild-type littermates. Surprisingly, the humoral response to DNP-Ficoll was significantly greater in CD19-/- mice. In contrast, hCD19TG mice were hyperresponsive to TNP-LPS and DNP-KLH immunization but were hyporesponsive to DNP-Ficoll. These results demonstrate that CD19 is not required for B-cell differentiation and isotype switching but serves as a response regulator which modulates B-cell differentiation. Since humoral responses to both T-cell-dependent and T-cell-independent antigens were similarly affected by alterations in CD19 expression, these differences are most likely to result from intrinsic changes in B-cell function rather than from the selective disruption of B-cell interactions with T cells.

Immunostimulatory oligodeoxynucleotides containing the CpG motif are effective as immune adjuvants in tumor antigen immunization

Recent advances in our understanding of the immune response are allowing for the logical design of new approaches to cancer immunization. One area of interest is the development of new immune adjuvants. Immunostimulatory oligodeoxynucleotides containing the CpG motif (CpG ODN) can induce production of a wide variety of cytokines and activate B cells, monocytes, dendritic cells, and NK cells. Using the 38C13 B cell lymphoma model, we assessed whether CpG ODN can function as immune adjuvants in tumor antigen immunization. The idiotype served as the tumor antigen. Select CpG ODN were as effective as complete Freund’s adjuvant at inducing an antigen-specific antibody response but were associated with less toxicity. These CpG ODN induced a higher titer of antigen-specific IgG2a than did complete Freund’s adjuvant, suggesting an enhanced TH1 response. Mice immunized with CpG ODN as an adjuvant were protected from tumor challenge to a degree similar to that seen in mice immunized with complete Freund’s adjuvant. We conclude that CpG ODN are effective as immune adjuvants and are attractive as part of a tumor immunization strategy.

An epidemic of tuberculosis with a high rate of tuberculin anergy among a population previously unexposed to tuberculosis, the Yanomami Indians of the Brazilian Amazon

A survey of an emerging tuberculosis epidemic among the Yanomami Indians of the Amazonian rain forest provided a unique opportunity to study the impact of tuberculosis on a population isolated from contact with the tubercle bacillus for millennia until the mid-1960s. Within the Yanomami population, an extraordinary high prevalence of active tuberculosis (6.4% of 625 individuals clinically examined) was observed, indicating a high susceptibility to disease, even among bacille Calmette–Guérin-vaccinated individuals. Observational studies on cell-mediated and humoral immune responses of the Yanomami Indians compared with contemporary residents of the region suggest profound differences in immunological responsiveness to Mycobacterium tuberculosis infection. Among the Yanomami, a very high prevalence of tuberculin skin test anergy was found. Of patients with active tuberculosis, 46% had purified protein derivative of tuberculosis reactions <10 mm; similarly 58% of recent bacillus Calmette–Guérin vaccines exhibited skin test reactions <5 mm. The Yanomami also had higher titers of antibodies against M. tuberculosis glycolipid antigens (>70%) than the control subjects comprised of Brazilians of European descent (14%). The antibodies were mostly of the IgM isotype. Among the tuberculosis patients who also produced IgG antibodies, the titers of IgG4 were significantly higher among the Yanomami than in the control population. Although it was not possible to analyze T-cell responses or patterns of lymphokine production in vitro because of the remoteness of the villages from laboratory facilities, the results suggest that the first encounter of the Yanomami Indian population with tuberculosis engenders a diminished cell-mediated immune response and an increased production antibody responses, relative to other populations with extensive previous contact with the pathogen. These findings suggest that tuberculosis may represent a powerful selective pressure on human evolution that over centuries has shaped the nature of human immune responses to infection.

Analysis of the Drosophila host defense in domino mutant larvae, which are devoid of hemocytes

We have analyzed the Drosophila immune response in domino mutant larvae, which are devoid of blood cells. The domino mutants have a good larval viability, but they die as prepupae. We show that, on immune challenge, induction of the genes encoding antimicrobial peptides in the fat body is not affected significantly in the mutant larvae, indicating that hemocytes are not essential in this process. The hemocoele of domino larvae contains numerous live microorganisms, the presence of which induces a weak antimicrobial response in the fat body. A full response is observed only after septic injury. We propose that the fat body cells are activated both by the presence of microorganisms and by injury and that injury potentiates the effect of microorganisms. Survival experiments after an immune challenge showed that domino mutants devoid of blood cells maintain a wild-type resistance to septic injury. This resistance was also observed in mutant larvae in which the synthesis of antibacterial peptides is impaired (immune deficiency larvae) and in mutants that are deficient for humoral melanization (Black cells larvae). However, if domino was combined with either the immune deficiency or the Black cell mutation, the resistance to septic injury was reduced severely. These results establish the relevance of the three immune reactions: phagocytosis, synthesis of antibacterial peptides, and melanization. By working in synergy, they provide Drosophila a highly effective defense against injury and/or infection.

Tumor cells expressing a retroviral envelope escape immune rejection in vivo

A model system for the in vivo control of tumor cell proliferation by the immune system has been used to assay for the possible immunosuppressive activity of retroviral proteins. Expression vectors for the entire or the transmembrane subunit of the Moloney murine leukemia virus envelope protein were constructed, as well as control vectors for irrelevant transmembrane proteins—or no protein. They were introduced either into MCA205 murine tumor cells, which do not proliferate upon s.c. injection into an allogeneic host, or into CL8.1 murine tumor cells, which overexpress class I antigens and are rejected in a syngeneic host. In both cases, expression of the complete envelope protein or of the transmembrane subunit resulted in tumor growth in vivo, with no effect of control vectors. Tumor cell growth results from inhibition of the host immune response, as the envelope-dependent effect was no more observed for MCA205 cells in syngeneic mice or for CL8.1 cells in x-irradiated mice. This inhibition is local because it is not observed at the level of control tumor cells injected contralaterally. These results suggest a noncanonical function of retroviral envelopes in the “penetrance” of viral infections, as well as a possible involvement of the envelope proteins of endogenous retroviruses in tumoral processes.

Autoimmune response to U1 small nuclear ribonucleoprotein (U1 snRNP) associated with cytomegalovirus infection

The induction of autoantibodies to U1 small nuclear ribonucleoprotein (U1 snRNP) complexes is not well understood. We present evidence that healthy individuals with cytomegalovirus (CMV) infection have an increased frequency and quantity of antibodies to ribonucleoprotein, directed primarily against the U1-70k protein. A significant association between the presence of antibodies to CMV and antibodies to the total RNP targeted by the immune response to the spliceosome (to both the Sm and RNP; Sm/RNP) was found for patients with systemic lupus erythematosus (SLE) but not those with mixed connective-tissue disease. CMV thus may play a role in inducing autoimmune responses in a subset of patients with systemic lupus erythematosus.

Increased sexual activity reduces male immune function in Drosophila melanogaster

Despite the benefits of resistance, susceptibility to infectious disease is commonplace. Although specific susceptibility may be considered an inevitable consequence of the co-evolutionary arms race between parasite and host, a more general constraint may arise from the cost of an immune response. This “cost” hypothesis predicts a tradeoff between immune defense and other components of fitness. In particular, a tradeoff between immunity and sexually selected male behavior has been proposed. Here we provide experimental support for the direct phenotypic tradeoff between sexual activity and immunity by studying the antibacterial immune response in Drosophila melanogaster. Males exposed to more females showed a reduced ability to clear a bacterial infection, an effect that we experimentally link to changes in sexual activity. Our results suggest immunosuppression is an important cost of reproduction and that immune function and levels of disease susceptibility will be influenced by sexual selection.

Genetically engineered poxviruses for recombinant gene expression, vaccination, and safety.

Vaccinia virus, no longer required for immunization against smallpox, now serves as a unique vector for expressing genes within the cytoplasm of mammalian cells. As a research tool, recombinant vaccinia viruses are used to synthesize and analyze the structure-function relationships of proteins, determine the targets of humoral and cell-mediated immunity, and investigate the types of immune response needed for protection against specific infectious diseases and cancer. The vaccine potential of recombinant vaccinia virus has been realized in the form of an effective oral wild-life rabies vaccine, although no product for humans has been licensed. A genetically altered vaccinia virus that is unable to replicate in mammalian cells and produces diminished cytopathic effects retains the capacity for high-level gene expression and immunogenicity while promising exceptional safety for laboratory workers and potential vaccine recipients.

Vaccination with syngeneic, lymphoma-derived immunoglobulin idiotype combined with granulocyte/macrophage colony-stimulating factor primes mice for a protective T-cell response.

The idiotype of the Ig expressed by a B-cell malignancy (Id) can serve as a unique tumor-specific antigen and as a model for cancer vaccine development. In murine models of Id vaccination, formulation of syngeneic Id with carrier proteins or adjuvants induces an anti-idiotypic antibody response. However, inducing a potent cell-mediated response to this weak antigen instead would be highly desirable. In the 38C13 lymphoma model, we observed that low doses of free granulocyte/macrophage colony-stimulating factor (GM-CSF) 10,000 units i.p. or locally s.c. daily for 4 days significantly enhanced protective antitumor immunity induced by s.c. Id-keyhole limpet hemocyanin (KLH) immunization. This effect was critically dependent upon effector CD4+ and CD8+ T cells and was not associated with any increased anti-idiotypic antibody production. Lymphocytes from spleens and draining lymph nodes of mice primed with Id-KLH plus GM-CSF, but not with Id-KLH alone, demonstrated significant proliferation to Id in vitro without any biased production of interferon gamma or interleukin 4 protein or mRNA. As a further demonstration of potency, 50% of mice immunized with Id-KLH plus GM-CSF on the same day as challenge with a large s.c. tumor inoculum remained tumor-free at day 80, compared with 17% for Id-KLH alone, when immunization was combined with cyclophosphamide. Taken together, these results demonstrate that GM-CSF can significantly enhance the immunogenicity of a defined self-antigen and that this effect is mediated exclusively by activating the T-cell arm of the immune response.

Induction of mucosal immune responses against a heterologous antigen fused to filamentous hemagglutinin after intranasal immunization with recombinant Bordetella pertussis.

Live vaccine vectors are usually very effective and generally elicit immune responses of higher magnitude and longer duration than nonliving vectors. Consequently, much attention has been turned to the engineering of oral pathogens for the delivery of foreign antigens to the gut-associated lymphoid tissues. However, no bacterial vector has yet been designed to specifically take advantage of the nasal route of mucosal vaccination. Herein we describe a genetic system for the expression of heterologous antigens fused to the filamentous hemagglutinin (FHA) in Bordetella pertussis. The Schistosoma mansoni glutathione S-transferase (Sm28GST) fused to FHA was detected at the cell surface and in the culture supernatants of recombinant B. pertussis. The mouse colonization capacity and autoagglutination of the recombinant microorganism were indistinguishable from those of the wild-type strain. In addition, and in contrast to the wild-type strain, a single intranasal administration of the recombinant strain induced both IgA and IgG antibodies against Sm28GST and against FHA in the bronchoalveolar lavage fluids. No anti-Sm28GST antibodies were detected in the serum, strongly suggesting that the observed immune response was of mucosal origin. This demonstrates, to our knowledge, for the first time that recombinant respiratory pathogens can induce mucosal immune responses against heterologous antigens, and this may constitute a first step toward the development of combined live vaccines administrable via the respiratory route.

Immunologic basis of transplant-associated arteriosclerosis.

Although immunosuppressive therapy minimizes the risk of graft failure due to acute rejection, transplant-associated arteriosclerosis of the coronary arteries remains a significant obstacle to the long-term survival of heart transplant recipients. The participation of specific inflammatory cell types in the genesis of this lesion was examined in a mouse model in which carotid arteries were transplanted across multiple histocompatibility barriers into seven mutant strains with immunologic defects. An acquired immune response--with the participation of CD4+ (helper) T cells, humoral antibody, and macrophages--was essential to the development of the concentric neointimal proliferation and luminal narrowing characteristic of transplant arteriosclerosis. CD8+ (cytotoxic) T cells and natural killer cells were not involved in the process. Arteries allografted into mice deficient in both T-cell receptors and humoral antibody showed almost no neointimal proliferation, whereas those grafted into mice deficient only in helper T cells, humoral antibody, or macrophages developed small neointimas. These small neointimas and the large neointimas of arteries grafted into control animals contained a similar number of inflammatory cells; however, smooth muscle cell number and collagen deposition were diminished in the small neointimas. Also, the degree of inflammatory reaction in the adventitia did not correlate with the size of the neointima. Thus, the reduction in neointimal size in arteries allografted into mice deficient in helper T cells, humoral antibody, or macrophages may be accounted for by a decrease in smooth muscle cell migration or proliferation.

The BALB/c mouse B-cell response to pigeon cytochrome c initiates as a heteroclitic response specific for the self antigen mouse cytochrome c.

Direct evidence is presented in support of the longstanding but unproven hypothesis that B lymphocytes specific for self antigens (Ags) can be used in the immune response to foreign Ags. We show that the B cells in BALB/c mic responding early to pigeon cytochrome c (CYT) produce antibodies that recognize and bind the major antigenic site on mouse CYT with greater affinity than they bind pigeon CYT i.e., they are heteroclitic for the self Ag. Furthermore, these B cells express the same combination of immunoglobulin variable region (V) genes that are known to be used in B-cell recognition of mouse CYT. Over time, the response to pigeon CYT becomes more specific for the foreign Ag through the recruitment of B cells expressing different combinations of V genes and, possibly, somatic mutation of the mouse CYT specific B cells from early in the response. Cross-recognition of pigeon CYT by mouse CYT-specific B cells results from the sharing of critical amino acid residues by the two Ags. Although B-cell recognition of the self Ag, mouse CYT, is very specific, which limits the extent to which foreign Ags can cross-activate the autoreactive B cells, it is possible that polyreactive B cells to other self Ags may be used more frequently in response to foreign Ags.