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Mycobacterium tuberculosis (Mtb) causes human tuberculosis, and more people die of it than of any other pathogen in the world. Immunodominant antigens elicit the large majority of T cells during an infection, making them logical vaccine candidates. Yet, it is still unknown whether these immunodominant antigen-specific T cells recognize Mtb-infected cells. Two immunodominant antigens, TB10.4 and Ag85b, have been incorporated into vaccine strategies. Surprisingly, mice vaccinated with TB10.4 generate TB10.4-specific memory CD8+ T cells but do not lead to additional protection compared to unvaccinated mice during TB. Ag85b-specific CD4+ T cells are also generated during vaccination, but the literature on whether these cells recognize Mtb-infected cells is also inconsistent. We demonstrate that TB10.4-specific CD8+ T cells do not recognize Mtb-infected cells. However, under the same conditions, Ag85b-specific CD4+ T cells recognize Mtb-infected macrophages and inhibit bacterial growth. In contrast, polyclonal CD4+ and CD8+ T cells from the lungs of infected mice can specifically recognize Mtb-infected macrophages, suggesting macrophages present antigens other than the immunodominant TB10.4. The antigen location may also be critical for presentation to CD8+ T cells, and live Mtb may inhibit antigen presentation of TB10.4. Finally, we propose that TB10.4 is a decoy antigen as it elicits a robust CD8+ T cell response that poorly recognizes Mtb-infected macrophages, allowing Mtb to evade host immunity.

Antígenos de alta massa molecular (PI) do extrato de Ascaris suum exercem efeito supressor sobre a resposta imune a antígenos heterólogos. PI atua diretamente sobre as DCs, diminuindo a expressão das moléculas coestimuladoras, MHC de classe II e assim, a proliferação de linfócitos T. Esse efeito é independente de TLR2, TLR4 e da molécula MyD88. Nesse trabalho estudamos a participação dos receptores DC-SIGN e MR, na modulação da atividade das DCs. PI contém oligossacarídeos N-ligados com cadeias de alta manose e do tipo complexa e contém resíduos de fosforilcolina. Os componentes do PI contendo as cadeias glicosídicas N-ligadas inibem a maturação de DCs incubadas com LPS. Receptores DC-SIGN e MR estão envolvidos no reconhecimento e internalização dos componentes do PI pelas DCs. O bloqueio desses receptores foi capaz de abolir o efeito inibitório do PI sobre as DCs e a resposta proliferativa de linfócitos T in vitro. Portanto, os resultados mostram a participação do DC-SIGN e MR no reconhecimento de componentes glicosilados do PI e na sua ação imunomoduladora. / High molecular weight components (PI) of Ascaris suum extract exert suppressive effect on the immune response to OVA. PI exert direct effect on DCs, decreasing the T lymphocyte proliferation. This effect is independent of TLR2 and 4 as well as MyD88 molecule. In this work we studied the glycoconjugates in PI and the participation of DC-SIGN and MR, in the modulation of the functional activity of DCs. PI components contain high mannose- and complex-type N-linked oligosaccharides and phosphorylcholine residues. PI components containing N-linked glycans inhibited the DCs maturation induced by LPS. The previous incubation of DCs with mannan, anti-DC-SIGN and anti-MR antibodies abolished the modulatory effect of PI on the DCs maturation. It was also observed that the blockage of DC-SIGN and MR in DCs reversed the inhibitory effect of PI in the in vitro T cells proliferative response. Taking together these results show the involvement of DC-SIGN and MR in the recognition of glycosylated components of PI by DCs and in its modulatory effect.

Antígenos de alta massa molecular (PI) do extrato de Ascaris suum exercem efeito supressor sobre a resposta imune a antígenos heterólogos. PI atua diretamente sobre as DCs, diminuindo a expressão das moléculas coestimuladoras, MHC de classe II e assim, a proliferação de linfócitos T. Esse efeito é independente de TLR2, TLR4 e da molécula MyD88. Nesse trabalho estudamos a participação dos receptores DC-SIGN e MR, na modulação da atividade das DCs. PI contém oligossacarídeos N-ligados com cadeias de alta manose e do tipo complexa e contém resíduos de fosforilcolina. Os componentes do PI contendo as cadeias glicosídicas N-ligadas inibem a maturação de DCs incubadas com LPS. Receptores DC-SIGN e MR estão envolvidos no reconhecimento e internalização dos componentes do PI pelas DCs. O bloqueio desses receptores foi capaz de abolir o efeito inibitório do PI sobre as DCs e a resposta proliferativa de linfócitos T in vitro. Portanto, os resultados mostram a participação do DC-SIGN e MR no reconhecimento de componentes glicosilados do PI e na sua ação imunomoduladora. / High molecular weight components (PI) of Ascaris suum extract exert suppressive effect on the immune response to OVA. PI exert direct effect on DCs, decreasing the T lymphocyte proliferation. This effect is independent of TLR2 and 4 as well as MyD88 molecule. In this work we studied the glycoconjugates in PI and the participation of DC-SIGN and MR, in the modulation of the functional activity of DCs. PI components contain high mannose- and complex-type N-linked oligosaccharides and phosphorylcholine residues. PI components containing N-linked glycans inhibited the DCs maturation induced by LPS. The previous incubation of DCs with mannan, anti-DC-SIGN and anti-MR antibodies abolished the modulatory effect of PI on the DCs maturation. It was also observed that the blockage of DC-SIGN and MR in DCs reversed the inhibitory effect of PI in the in vitro T cells proliferative response. Taking together these results show the involvement of DC-SIGN and MR in the recognition of glycosylated components of PI by DCs and in its modulatory effect.

T cell receptors (TCR) bind to peptides from various sources on MHC (Major Histocompatibility Complex) molecules. A long-standing goal in the field is to understand the mechanisms of MHC-peptide exchange and MHC-TCR interactions. Here, I present work from three uniquely different systems that address the following: HLA-DR1 conformational stability, self-tolerant mechanisms of TCRs isolated from self-reactive TCR transgenic mice, and TCR cross-reactivity mechanisms between LCMV and VV. First, I present a crystal structure of HLA-DR1 in complex with A1L9 peptide, a peptide with two amino acid substitutions from the parental peptide. The singly substituted A1 peptide, which has a pocket 1 alanine substitution, decreases intrinsic half-life between MHC-peptide and increases susceptibility to HLA-DM mediated peptide exchange. This data agrees with previous models of HLA-DM-mediated peptide exchange in which the major determinant is located at the HLA-DR1 pocket 1. However, the L9 substituted peptide, which has a pocket 9 leucine substitution, displays the opposite phenotype: increased intrinsic half-life and decreased HLA-DM susceptibility. The crystal structure presented here shows that HLA-DR1 in complex with a doubly substituted peptide, A1L9, is in the same conformation as HLA-DR1 with the wild-type peptide, demonstrating that pocket 9 residues can rescue pocket 1 residue binding deficiencies and that HLA-DR1 stability is determined by amino acids along the peptide, not only at pocket 1. Next, I present crystal structures of two self-tolerant TCRs in complex with IAb-3K pMHC. To elucidate molecular mechanism for self-reactivity and self-tolerance, the TCRs J809.B5 and 14.C6 are compared to each other and its parental self-reactive TCR, YAe-62.8. In comparison to YAe-62.8, J809.B5 interacts with the same pMHC, but utilizes more peptide specific interactions, a mechanism that may distinguish self-reactive receptors from self-tolerant receptors. Additionally, the crystal structure of 14.C6 TCR, which bears a different CDR3α sequence from J809.B5, demonstrates that CDR3 sequences can modulate interactions of germline encoded CDR1 and CDR2 loops. Together, these results highlight that in addition to CDR3 VDJ recombination, diversity is generated in the mature TCR repertoire by differential chain pairing, either of which can affect the interactions of germline encoded CDR loops. Next, I present a detailed analysis of cross-reactive TCRs between Kb-GP34 and Kb-A11R. The mature LCMV-immune repertoire was analyzed by DNA deep sequencing of TCRβ CDR3 sequences, which led to the identification of new cross-reactive sequence motifs. Cross-reactive sequence motifs varied by each Vβ gene, suggesting a role of CDR1, CDR2, and CDR3 loop interplay in cross-reactivity. Lastly, I present the crystal structures of a GP34/A11R cross-reactive TCR in complex with both Kb-GP34 and Kb-A11R. Analysis of the crystal structures revealed that the two complexes are largely the same, despite differences in peptide sequences. Surprisingly, the TCR to peptide interactions were dominated by three out of eight peptide side-chains. Cross-reactivity between these two complexes is likely due to a large amount of interactions from TCR to MHC compared to interactions of TCR to peptide. We note two unique MHC-peptide interactions that may allow Kb to be an allele prone to cross-reactivity. The first is an interaction at the C-terminus of the A11R peptide which pulls A11R P7 asparagine away from TCR interactions. The second interaction is from an arginine at position 155, which sits at the interface between TCRα and TCRβ , and contributes the most buried surface area in the interaction interface. Because Kb’s arginine 155 is a long side chain that hydrogen bonds with the peptide backbone, and is also at the center of the TCR-peptide interface, GP34 and A11R peptide sequence differences may be occluded from TCR discrimination by Kb presentation. The data presented in this dissertation demonstrate that interactions between MHC-peptide and MHC-TCR act harmoniously and coopertively, whereby proximal interactions are affected by interactions elsewhere. While previous models of HLA-DR/HLA-DM interactions demonstrate the importance of interactions at HLA-DR1 pocket 1, I showed that pocket 9 also contributes to HLA-DR stability and therefore, HLA-DM susceptibility. I also showed that TCR CDR3 loop sequences affect germline CDR1/CDR2 loop interactions and vice versa. Lastly, I showed that allele specific MHC side chain interactions with the bound peptide influence TCR ligand binding and hence, TCR cross-reactivity.