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In this work, we studied autoantibody repertoires and Ig isotypes in 71 mothers and their 104 healthy newborns (including twins and triplets delivered term or premature). Newborns receive maternal IgG Abs via the placenta before birth, but developing infants must produce their own IgM and IgA Abs. We used an Ag microarray analysis to detect binding to a selection of 295 self-Ags, compared with 27 standard foreign Ags. The magnitude of binding to specific self-Ags was found to be not less than that to the foreign Ags. As expected, each newborn shared with its mother a similar IgG repertoire—manifest as early as the 24th week of gestation. IgM and IgA autoantibody repertoires in cord sera were highly correlated among the newborns and differed from their mothers’ repertoires; the latter differed in sera and milk. The autoantibodies bound to self-Ags known to be associated with tumors and to autoimmune diseases. Thus, autoantibody repertoires in healthy humans—the immunological homunculus—arise congenitally, differ in maternal milk and sera, and mark the potential of the immune system to attack tumors, beneficially, or healthy tissues, harmfully; regulation of the tissue site, the dynamics, and the response phenotype of homuncular autoimmunity very likely affects health.

Gliomas that grow uninhibited in the brain almost never metastasize outside the CNS. The rare occurrences of extracranial metastasis are usually associated with a suppressed immune system. This observation raises the possibility that some gliomas might not grow outside the CNS due to an inherent immune response, We report in this study that the highly malignant F98 Fischer rat undifferentiated glioma, which grows aggressively in the brain, spontaneously regresses when injected live s.c. We found that this regression is immune-mediated and that it markedly enhances the survival or cures rats challenged with the same tumor intracranially either before or after the s.c. live-cell treatment. Adoptive transfer experiments showed the effect was immune-mediated and that the CD8 T cell fraction, which exhibited direct tumor cytotoxicity, was more effective than the CD4 T cell fraction in mediating resistance to intracranial challenge of naive rats. Brain tumors from treated rats exhibited enhanced CD3+CD8+CD4− and CD3+CD4+CD8− T cell infiltration and IFN-γ secretion. The results in the F98 glioma were corroborated in the Lewis rat CNS-1 astrocytoma. In both tumor models, s.c. treatment with live cells was significantly better than immunization with irradiated cells. We propose in this study a location-based immunotherapeutic phenomenon we term “split immunity”: a tumor that thrives in an immune-privileged site may be inhibited by injecting live, unmodified tumor cells into a site that is not privileged, generating protective immunity that spreads back to the privileged site. Split immunity could explain several long-standing paradoxes regarding the lack of overt extracranial metastasis in patients with primary brain tumors.

LPS, a molecule produced by Gram-negative bacteria, is known to activate both innate immune cells such as macrophages and adaptive immune B cells via TLR4 signaling. Although TLR4 is also expressed on T cells, LPS was observed not to affect T cell proliferation or cytokine secretion. We now report, however, that LPS can induce human T cells to adhere to fibronectin via TLR4 signaling. This response to LPS was confirmed in mouse T cells; functional TLR4 and MyD88 were required, but T cells from TLR2 knockout mice could respond to LPS. The human T cell response to LPS depended on protein kinase C signaling and involved the phosphorylation of the proline-rich tyrosine kinase (Pyk-2) and p38. LPS also up-regulated the T cell expression of suppressor of cytokine signaling 3, which led to inhibition of T cell chemotaxis toward the chemokine stromal cell-derived factor 1α (CXCL12). Thus, LPS, through TLR4 signaling, can affect T cell behavior in inflammation.

Extracellular heat shock protein 60 (HSP60) has been considered a proinflammatory danger signal. Yet, HSP60 can also down-regulate experimental immune arthritis and diabetes models by specific inhibition of Th1-like responses. We now report that HSP60 in vitro differentially modulates the expression of Th1/Th2 transcription factors in human T cells: HSP60 down-regulates T-bet, NF-κB, and NFATp and up-regulates GATA-3, leading to decreased secretion of TNF-α and IFN-γ and enhanced secretion of IL-10. These effects depended on TLR2 signaling and could not be attributed to LPS or to other contaminants. In BALB/c mice, HSP60 in vivo inhibited the clinical, histological, and serological manifestations of Con A-induced hepatitis associated with up-regulated T cell expression of suppressor of cytokine signaling 3 and GATA-3 and down-regulated T-bet expression. These results provide a molecular explanation for the effects of HSP60 treatment on T cell inflammation via innate regulation of the inflammatory response.

Vascular endothelial growth factor (VEGF) and its receptors are critical in angiogenesis. The main player in the secretion and response to VEGF is the endothelial cell. We initiated this study to test whether T cells can secrete VEGF and are able to respond to it. Here we show that VEGF is secreted by T cells on stimulation by specific Ag or by IL-2 and by hypoxia; thus, activated T cells might enhance angiogenesis. Hypoxia also induced the expression in T cells of VEGFR2, suggesting that T cells might also respond to VEGF. Indeed, VEGF augmented IFN-γ and inhibited IL-10 secretion by T cells responding to mitogen or Ag; thus, VEGF can enhance a Th1 phenotype. Encephalitogenic T cells stimulated in the presence of VEGF caused more severe and prolonged encephalomyelitis. Thus, T cells can play a role in angiogenesis by delivering VEGF to inflammatory sites, and VEGF can augment proinflammatory T cell differentiation.

Adjuvant arthritis (AA) is induced by immunizing Lewis rats with Mycobacterium tuberculosis suspended in adjuvant. The mycobacterial 65-kDa heat shock protein (HSP65) contains at least one epitope associated with the pathogenesis of AA: T cell clones that recognize an epitope formed by aa 180–188 of HSP65 react with self-cartilage and can adoptively transfer AA. Nevertheless, vaccination with HSP65 or some of its T cell epitopes can prevent AA by a mechanism that seems to involve cross-reactivity with the self-60-kDa HSP60. We recently demonstrated that DNA vaccination with the human hsp60 gene can inhibit AA. In the present work, we searched for regulatory epitopes using DNA vaccination with HSP60 gene fragments. We now report that specific HSP60 DNA fragments can serve as effective vaccines. Using overlapping HSP60 peptides, we identified a regulatory peptide (Hu3) that was specifically recognized by the T cells of DNA-vaccinated rats. Vaccination with Hu3, or transfer of splenocytes from Hu3-vaccinated rats, inhibited the development of AA. Vaccination with the mycobacterial homologue of Hu3 had no effect. Effective DNA or peptide vaccination was associated with enhanced T cell proliferation to a variety of disease-associated Ags, along with a Th2/3-like shift (down-regulation of IFN-γ secretion and enhanced secretion of IL-10 and/or tumor growth factor β1) in response to peptide Mt176–190 (the 180–188 epitope of HSP65). The regulatory response to HSP60 or its Hu3 epitope included both Th1 (IFN-γ) and Th2/3 (IL-10/tumor growth factor β1) secretors. These results show that regulatory mechanisms can be activated by immunization with relevant self-HSP60 epitopes.

We induced T cell help for vaccination against Streptococcus pneumoniae (Pn) using self and foreign peptides and their source proteins conjugated to the capsular polysaccharide (CPS) of type 4 Pn; the carriers were self-heat shock protein 60 (HSP60) and tetanus toxoid (TT). We measured the production of IgG Abs to the CPS and the carriers, and tested resistance to challenge with highly lethal amounts of Pn injected i.p. (LD50 × 103–106). We now report that vaccination protects old and young mice from bacterial challenge; however, there were significant differences in vaccine efficacy based on the carrier. Self-HSP60 peptide p458m was more effective than the whole HSP60 molecule and was equally effective compared with TT. Both p458m and TT were more protective than the TT-derived peptide p30 after a single vaccination. However, peptide p30 was effective in more MHC genotypes than was p458m. Unlike other vaccines, protection conferred by p458m was not related to the amount of anti-CPS Ab: mice that produced very little Ab were still protected from highly lethal doses of bacteria (LD50 × 105–106). Furthermore, unlike the other carriers, there was no Ab response to the p458m carrier. Thus, peptides, self as well as foreign, can provide T cell help that differs functionally from that provided by the whole parent protein.

Nonobese diabetic (NOD) mice spontaneously develop diabetes as a consequence of an autoimmune process that can be inhibited by immunotherapy with the 60-kDa heat shock protein (hsp60), with its mycobacterial counterpart 65-kDa (hsp65), or with other Ags such as insulin and glutamic acid decarboxylase (GAD). Microbial infection and innate signaling via LPS or CpG motifs can also inhibit the spontaneous diabetogenic process. In addition to the spontaneous disease, however, NOD mice can develop a more robust cyclophosphamide-accelerated diabetes (CAD). In this work, we studied the effect on CAD of DNA vaccination with constructs encoding the Ags human hsp60 (phsp60) or mycobacterial hsp65 (phsp65). Vaccination with phsp60 protected NOD mice from CAD. In contrast, vaccination with phsp65, with an empty vector, or with a CpG-positive oligonucleotide was not effective, suggesting that the efficacy of the phsp60 construct might be based on regulatory hsp60 epitopes not shared with its mycobacterial counterpart, hsp65. Vaccination with phsp60 modulated the T cell responses to hsp60 and also to the GAD and insulin autoantigens; T cell proliferative responses were significantly reduced, and the pattern of cytokine secretion to hsp60, GAD, and insulin showed an increase in IL-10 and IL-5 secretion and a decrease in IFN-γ secretion, compatible with a shift from a Th1-like toward a Th2-like autoimmune response. Our results extend the role of specific hsp60 immunomodulation in the control of β cell autoimmunity and demonstrate that immunoregulatory networks activated by specific phsp60 vaccination can spread to other Ags targeted during the progression of diabetes, like insulin and GAD.

Adjuvant arthritis (AA) is an autoimmune disease inducible in rats involving T cell reactivity to the mycobacterial 65-kDa heat shock protein (HSP65). HSP65-specific T cells cross-reactive with the mammalian 60-kDa heat shock protein (HSP60) are thought to participate in the modulation of AA. In this work we studied the effects on AA of DNA vaccination using constructs coding for HSP65 (pHSP65) or human HSP60 (pHSP60). We found that both constructs could inhibit AA, but that pHSP60 was more effective than pHSP65. The immune effects associated with specific DNA-induced suppression of AA were complex and included enhanced T cell proliferation to a variety of disease-associated Ags. Effective vaccination with HSP60 or HSP65 DNA led paradoxically to up-regulation of IFN-γ secretion to HSP60 and, concomitantly, to down-regulation of IFN-γ secretion to the P180-188 epitope of HSP65. There were also variable changes in the profiles of IL-10 secretion to different Ags. However, vaccination with pHSP60 or pHSP65 enhanced the production of TGFβ1 to both HSP60 and HSP65 epitopes. Our results support a regulatory role for HSP60 autoreactivity in AA and demonstrate that this control mechanism can be activated by DNA vaccination with both HSP60 or HSP65.

Nonobese diabetic (NOD) mice develop insulitis and diabetes through a process involving autoimmunity to the 60-kDa heat shock protein (HSP60). Treatment of NOD mice with HSP60 or with peptides derived from HSP60 inhibits this diabetogenic process. We now report that NOD diabetes can be inhibited by vaccination with a DNA construct encoding human HSP60, with the pcDNA3 empty vector, or with an oligonucleotide containing the CpG motif. Prevention of diabetes was associated with a decrease in the degree of insulitis and with down-regulation of spontaneous proliferative T cell responses to HSP60 and its peptide p277. Moreover, both the pcDNA3 vector and the CpG oligonucleotide induced specific Abs, primarily of the IgG2b isotype, to HSP60 and p277, and not to other islet Ags (glutamic acid decarboxylase or insulin) or to an unrelated recombinant Ag expressed in bacteria (GST). The IgG2b isotype of the specific Abs together with the decrease in T cell proliferative responses indicate a shift of the autoimmune process to a Th2 type in treated mice. These results suggest that immunostimulation by bacterial DNA motifs can modulate spontaneous HSP60 autoimmunity and inhibit NOD diabetes.

We investigated the interaction between T lymphocytes and macrophages in the in vitro sensitization of lymphocytes against tumor cells. Spleen cells were sensitized in vitro by syngeneic peritoneal macrophages that had been fed with cell-free antigen preparation of syngeneic tumor cells. The sensitized T lymphocytes acquired specific cytotoxic activity in vitro and the capacity to inhibit tumor development in vivo. Allogeneic macrophages, syngeneic fibroblasts, or the antigen preparation by itself were not able to sensitize the lymphocytes against the tumor.

We have shown previously that initiator T lymphocytes (ITL), sensitized in vitro against fibroblast antigens, recruit effector T cells in vivo. After injection into hind footpads of syngeneic recipients, sensitized ITL migrated to the draining popliteal lymph nodes (PLN) and activated a trapping mechanism by which circulating lymphocytes were recruited in the PLN. This paper reports experiments designed to test the immunospecificity of these recruited T lymphocytes (RTL). We found that immunospecific RTL were depleted from other lymphoid organs during recruitment in the PLN. However, immunospecific ITL were not depleted from spleens during PLN recruitment. Thus ITL and RTL are functionally distinguishable. We show that specific GVH reactive lymphocytes were also lost from spleens and distal lymph nodes during trapping of RTL in the PLN. Thus, the trapping phase of the recruitment response is immunospecific, as are the sensitization and effector phases. The trapped RTL are antigen-specific, and include the pool of GVH-reactive-lymphocytes committed to the same alloantigen. Thus, it appears that GVH-reactive cells respond to syngeneic ITL sensitized against allogeneic fibroblasts.

Summary A study was made of the resistance of germfree and conventional mice to direct challenge with Group A streptococci of graded virulence. Germ-free and conventional mice were highly, and equally, resistant to nonencapsulated strains of Group A streptococci lacking M protein. Greater susceptibility of germ-free mice could be demonstrated only with Group A strains containing both M protein and capsules. This difference could be abolished by a globulin fraction of normal mouse serum but not by normal mouse γ-globulin or by homologous type-specific antiserum. In vitro studies with bloods of germ-free mice and colostrum-deprived piglets provided additional evidence that phagocytosis of avirulent Group A streptococci required little, if any, specific antibodies. Bloods from these animals phagocytized and destroyed streptococci as readily as did their conventional counterparts. Thermolabile opsonins against avirulent Group A streptococci were readily demonstrable in the blood of colostrum-deprived piglets, whose serum contains only minute amounts of γ-globulin. Absorption of the sera of these animals at low temperatures with large amounts of homologous streptococci resulted in some decrease in opsonization, but considerable residual phagocytosis was apparent. The leukocytes of colostrum-deprived, and of colostrum-fed, animals were equally capable of phagocytizing and destroying Group A streptococci in the presence or absence of specific opsonins.

Summary Natural human 19S (IgM) and 7S (IgG) antibodies reactive with Neisseria gonorrhoeae were assayed by agglutination, bactericidal and indirect fluorescent antibody (IFA) procedures. Fluorescent antibody reagents specific for IgG and IgM were used in the IFA procedure. It was found that purified 19S serum fractions contained antibodies which were readily detected by agglutination, bactericidal and IFA techniques using an anti-IgM reagent. However, bactericidal and agglutination tests appeared to be relatively insensitive to natural IgG antibodies in purified 7S fractions. In contrast, the indirect fluorescent antibody technique using an anti-IgG reagent could detect high titers of IgG antibodies within the 7S fractions. A prozone phenomenon was observed in the IFA reactivity of IgM antibodies in whole serum. It was found that the addition of purified 7S antibodies to purified 19S antibody fractions decreased the ability of the 19S fraction to react with the anti-IgM reagent. This suggested that IgG antibodies inhibited IgM antibody reactivity in the IFA procedure. A partial inhibition of 19S bactericidal activity by 7S fractions was also found. This study indicates that the unequal capacity of different antibody classes to produce reactions, and the interactions between antibodies themselves in various test systems, must be taken into account in the interpretation of the results of immunologic tests.