Xavier Martinez

Influence of maternal antibodies on vaccine responses: inhibition of antibody but not T cell responses allows successful early prime‐boost strategies in mice

The transfer of maternal antibodies to the offspring and their inhibitory effects on active infant immunization is an important factor hampering the use of certain vaccines, such as measles or respiratory syncytial virus vaccine, in early infancy. The resulting delay in protection by conventional or novel vaccines may have significant public health consequences. To define immunization approaches which may circumvent this phenomenon, experiments were set up to further elucidate its immunological bases. The influence of maternal antibodies on antibody and T cell responses to measles hemagglutinin (MV‐HA) were analyzed following MV‐HA immunization of pups born to immune or control BALB/c mothers using four different antigen delivery systems: live or inactivated conventional measles vaccine, a live recombinant canarypox vector and a DNA vaccine. High levels (> 5 log10) of maternal anti‐HA antibodies totally inhibited antibody responses to each of the vaccine constructs, whereas normal antibody responses were elicited in presence of lower titers of maternal antibodies. However, even high titers of maternal antibodies affected neither the induction of vaccine‐specific Th1/Th2 responses, as assessed by proliferation and levels of IFN‐γ and IL‐5 production, nor CTL responses in infant mice. On the basis of these unaltered T cell responses, very early priming and boosting (at 1 and 3 weeks of age, respectively) with live measles vaccine allowed to circumvent maternal antibody inhibition of antibody responses in pups of immune mothers. This was confirmed in another immunization model (tetanus toxoid). It suggests that effective vaccine responses may be obtained earlier in presence of maternal antibodies through the use of appropriate immunization strategies using conventional or novel vaccines for early priming.

Induction of neonatal TH1 and CTL responses by live viral vaccines: a role for replication patterns within antigen presenting cells?

Failure to generate CTL responses in early life has been linked to the preferential maturation of CD4 T cells into TH2 rather than TH1 cells in response to some, but not other, antigenic stimulations. Here, we provide preliminary evidence for the role of the viral replication pattern in the shaping of neonatal cellular responses to live viral vaccines. Neonatal and early life immunization with live attenuated Sendai virus vaccine led to the induction of IgG2a antibodies and cytotoxic responses as efficiently as immunization of adult animals. Similarly, although early life immunization with live attenuated measles virus led to preferential TH2 polarization of T cells compared with adult primed animals, it allowed the induction of CTL responses which had not been observed following immunization with a live recombinant canarypox vector. Thus, conversely to a non-replicating canarypox recombinant vaccine expressing the measles haemagglutonin, viral vaccines with limited but present replication capacity appear capable of activating neonatal antigen presenting cells to trigger TH1 and CTL responses, as recently observed for DNA vaccines.

Combining DNA and protein vaccines for early life immunization against respiratory syncytial virus in mice

Early life responses to respiratory syncytial virus (RSV)‐F DNA and RSV‐F protein immunization were studied in murine models of neonatal immunization. RSV‐F DNA induced similar antibody (Ab) responses, antigen‐specific IFN‐γ production and cytotoxic T lymphocyte (CTL) responses in 1‐week‐old and adult BALB / c mice. In contrast, RSV‐F protein induced much higher IL‐5 responses in early life. Both vaccines elicited Ab and CTL responses in spite of maternal Ab, but with distinctive kinetics. Sequential RSV‐F DNA priming / protein boosting primed 1‐week‐old mice for RSV‐F‐specific CTL responses, reduced IL‐5 production and enhanced Ab responses. In contrast, IL‐5 exceeded IFN‐γ responses when young mice were primed with protein and boosted with DNA. Last, when protein and DNA immunization were combined, a single vaccine dose induced early Ab responses, preferential IL‐5 responses but strong CTL responses. Sequential or combined DNA / protein immunization thus represent interesting strategies for early life immunization.

CD8+ T Cell Tolerance in Nonobese Diabetic Mice Is Restored by Insulin-Dependent Diabetes Resistance Alleles

Although candidate genes controlling autoimmune disease can now be identified, a major challenge that remains is defining the resulting cellular events mediated by each locus. In the current study we have used NOD-InsHA transgenic mice that express the influenza hemagglutinin (HA) as an islet Ag to compare the fate of HA-specific CD8+ T cells in diabetes susceptible NOD-InsHA mice with that observed in diabetes-resistant congenic mice having protective alleles at insulin-dependent diabetes (Idd) 3, Idd5.1, and Idd5.2 (Idd3/5 strain) or at Idd9.1, Idd9.2, and Idd9.3 (Idd9 strain). We demonstrate that protection from diabetes in each case is correlated with functional tolerance of endogenous islet-specific CD8+ T cells. However, by following the fate of naive, CFSE-labeled, islet Ag-specific CD8+ (HA-specific clone-4) or CD4+ (BDC2.5) T cells, we observed that tolerance is achieved differently in each protected strain. In Idd3/5 mice, tolerance occurs during the initial activation of islet Ag-specific CD8+ and CD4+ T cells in the pancreatic lymph nodes where CD25+ regulatory T cells (Tregs) effectively prevent their accumulation. In contrast, resistance alleles in Idd9 mice do not prevent the accumulation of islet Ag-specific CD8+ and CD4+ T cells in the pancreatic lymph nodes, indicating that tolerance occurs at a later checkpoint. These results underscore the variety of ways that autoimmunity can be prevented and identify the elimination of islet-specific CD8+ T cells as a common indicator of high-level protection.

Expression of Diabetes-Associated Genes by Dendritic Cells and CD4 T Cells Drives the Loss of Tolerance in Nonobese Diabetic Mice

In humans and NOD mice, defects in immune tolerance result in the spontaneous development of type-1-diabetes. Recent studies have ascribed a breakdown in tolerance to dysfunction in regulatory T cells that is secondary to reduced IL-2 production by T cells having the NOD diabetes susceptibility region insulin-dependent diabetes 3 (Idd3). In this study, we demonstrate a peripheral tolerance defect in the dendritic cells of NOD mice that is independent of regulatory T cells. NOD CD8 T cells specific for islet Ags fail to undergo deletion in the pancreatic lymph nodes. Deletion was promoted by expression of the protective alleles of both Idd3 (Il2) and Idd5 in dendritic cells. We further identify a second tolerance defect that involves endogenous CD4 T cell expression of the disease-promoting NOD alleles of these genetic regions. Pervasive insulitis can be reduced by expression of the Idd3 and Idd5 protective alleles by either the Ag-presenting cell or lymphocytes.

Idd9.2 and Idd9.3 Protective Alleles Function in CD4+ T-Cells and Nonlymphoid Cells to Prevent Expansion of Pathogenic Islet-Specific CD8+ T-Cells

OBJECTIVE Multiple type 1 diabetes susceptibility genes have now been identified in both humans and mice, yet mechanistic understanding of how they impact disease pathogenesis is still minimal. We have sought to dissect the cellular basis for how the highly protective mouse Idd9 region limits the expansion of autoreactive CD8+ T-cells, a key cell type in destruction of the islets. RESEARCH DESIGN AND METHODS We assess the endogenous CD8+ T-cell repertoire for reactivity to the islet antigen glucose-6-phosphatase–related protein (IGRP). Through the use of adoptively transferred T-cells, bone marrow chimeras, and reconstituted severe combined immunodeficient mice, we identify the protective cell types involved. RESULTS IGRP-specific CD8+ T-cells are present at low frequency in the insulitic lesions of Idd9 mice and could not be recalled in the periphery by viral expansion. We show that Idd9 genes act extrinsically to the CD8+ T-cell to prevent the massive expansion of pathogenic effectors near the time of disease onset that occurs in NOD mice. The subregions Idd9.2 and Idd9.3 mediated this effect. Interestingly, the Idd9.1 region, which provides significant protection from disease, did not prevent the expansion of autoreactive CD8+ T-cells. Expression of Idd9 genes was required by both CD4+ T-cells and a nonlymphoid cell to induce optimal tolerance. CONCLUSIONS Idd9 protective alleles are associated with reduced expansion of IGRP-specific CD8+ T-cells. Intrinsic expression of protective Idd9 alleles in CD4+ T-cells and nonlymphoid cells is required to achieve an optimal level of tolerance. Protective alleles in the Idd9.2 congenic subregion are required for the maximal reduction of islet-specific CD8+ T-cells.

The Use of Idd Congenic Mice to Identify Checkpoints of Peripheral Tolerance to Islet Antigen

Abstract:  Type 1 diabetes (T1D) occurs because of lack of T cell tolerance to islet antigens. We hypothesized that critical genetic susceptibility loci that control progression to T1D, designated as insulin‐dependent diabetes (Idd) loci, would be responsible for preventing CD8 T cell tolerance. To test this hypothesis, we have used two different congenic non‐obese diabetic (NOD) mice that are highly protected from the occurrence of T1D because they express protective alleles at Idd3 and Idd5.1, 5.2, 5.3 (Idd3/5 mice), or at Idd9.1, 9.2, and 9.3 (Idd9 mice). By examining the CD8 T response to two different islet‐expressed antigens, we have determined that CD8 T tolerance is restored in both strains of mice. However, tolerance occurs at different checkpoints in each strain. In Idd3/5 mice, islet‐antigen‐specific CD8 T cells are eliminated in the pancreatic lymph nodes, where they are first activated by cross‐presented islet antigens. In contrast, in Idd9 mice autoreactive CD8 T cells accumulate at this site and are not tolerized until after they enter the pancreas. We are currently identifying the cell types and mechanisms that are critical for tolerance induction at each checkpoint.

Partial activation of neonatal CD11c+ dendritic cells and induction of adult-like CD8+ cytotoxic T cell responses by synthetic microspheres

Neonatal cytotoxic T cell responses have only been elicited to date with immunogens or delivery systems inducing potent direct APC activation. To define the minimal activation requirements for the induction of neonatal CD8+ cytotoxic responses, we used synthetic microspheres (MS) coated with a single CD8+ T cell peptide from lymphocytic choriomeningitis virus (LCMV) or HIV-1. Unexpectedly, a single injection of peptide-conjugated MS without added adjuvant induced CD4-dependent Ag-specific neonatal murine cytotoxic responses with adult-like CTL precursor frequency, avidity for Ag, and frequency of IFN-γ-secreting CD8+ splenocytes. Neonatal CD8+ T cell responses to MS-LCMV were elicited within 2 wk of a single immunization and, upon challenge, provided similar protection from viral replication as adult CTLs, demonstrating their in vivo competence. As previously reported, peptide-coated MS elicited no detectable activation of adult CD11c+ dendritic cells (DC). In contrast, CTL responses were associated with a partial activation of neonatal CD11c+ DC, reflected by the up-regulation of CD80 and CD86 expression but no concurrent changes in MHC class II or CD40 expression. However, this partial activation of neonatal DC was not sufficient to circumvent the requirement for CD4+ T cell help. The effective induction of neonatal CD8+ T cell responses by this minimal Ag delivery system demonstrates that neonatal CD11c+ DC may mature sufficiently to stimulate naive CD8+ neonatal T cells, even in the absence of strong maturation signals.