T cell selection Flashcards
evidence that expression of B chain prevents further B chain rearrangement
EXPERIMENTAL: Uematsu et al (1988)
Evidence for importance of expressed β chain in suppressing further β-locus rearrangement
Produced transgenic mice expressing rearranged β chain transgene
FACS = verified that almost all T lymphocytes in mice expressed transgenic β chain
Northern blotting = revealed transcription of transgene was largely T cell-specific
Cytolytic experiments = revealed that T lymphocytes in transgenic mice only express transgenic β chains, and not endogenous chains
- used monoclonal antibodies to selectively block transgenic β chain of T cells isolated from lymph nodes of transgenic and WT mice then stimulated in vitro
- T cells from transgenic mice showed no cytolytic activity in presence of antibodies
- Addition of antibodies to T cells from WT mice made no difference to cytolysis
Expression of transgenic β chain prevented complete rearrangement of endogenous β chains = only partial Dβ1-Jβ1 rearrangements and no endogenous VDJ rearrangements revealed by Southern blotting
principle of MHC restriction
Zinkernagel bone marrow chimera experiments: demonstrated principle of MHC-restriction
• Irradiated mice that possessed distinct MHC haplotypes (H-2k and H-2b) and transplanted with haematopoietic stem cells expressing both haplotypes (H-2bxk)
• Isolated mature T cells from thymus and tested ability to respond to antigen in context of each MHC molecule in vitro
• Despite all T cells deriving from same precursor population, adult T cells could only respond to antigens presented on antigen-presented cells expressing the same MHC expressed in thymus first evidence that T cells undergo selection process dependent on TCR-MHC interactions
evidence for role of TCR activation in positive selection
Takahama et al (1994)
- Provided further evidence of the role of TCR activation during positive selection
- Thymocytes from an MHC -/- mouse line were cultured in foetal thymic organ cultures (FTOCs) and antibodies against CD4 and CD8 revealed an absence of single-positive cells, as expected
- However, addition of anti-TCR monoclonal antibodies capable of inducing TCR dimerization permitted the generation of CD4+CD8- T-cells, showing that TCR dimerization is sufficient for the positive selection of CD4+ cells
- Much less able to promote formation of CD8+ CD4- cells = failed to explore why
evidence that self-peptides are important in positive selection
Nikolic-Zugic et al (1990) = EVIDENCE THAT SELF-PEPTIDES ARE IMPORTANT IN POSITIVE SELECTION
• Produced strains of mice with mutations in the peptide binding site of H-2kb class I MHC molecule
• Thymocytes developing in a WT thymus gave rise to a repertoire that could respond to immunogenic portion of foreign peptide ovalbumin
• T cells that had developed in mutant mice could not respond to this epitope
• Nature of peptides bound to MHC molecules has a direct impact on specificity of T cell repertoire
evidence for stochastic model of preceptor selection + critique
EXPERIMENTAL EVIDENCE: Itano et al (1994) = evidence for stochastic selection of CD8 • Compared phenotype of thymocytes from transgenic mice expressing F5 TCR (MHC class I restricted), and transgenic mice expressing F5 TCR and constitutive CD8.1 transgene • Found that constitutive expression of CD8 during thymic selection permit development of mature CD4+ cells bearing class I-restricted F5 TCR • Flow cytometry revealed that double transgenic mice contained significant proportion of T cells positive for endogenous CD4 and negative for endogenous CD8, seen to a much lesser degree in F5 TCR transgenic mice • Insertion of transgenic CD8 coreceptor is hence sufficient to rescue population of MHC class I specific T cells expressing endogenous CD4 from apoptosis • Problems with this model = substantial number of mismatched thymocytes not rescued by constitutive coreceptor expression = still a bias towards CD8+ cells in periphery = stochastic pathway is likely only a minor pathway
evidence that coreceptor expression dependent on TCR kinetics
• EXP: Yasutomo et al (2000) = distinct TCR signalling kinetics influence lineage selection
- Longer or shorter exposure to strong TCR ligands in cultured thymocytes induces development to CD4 or CD8 lineage respectively
evidence that coreceptor expression dependent on Lck
• EXP: Hernandez-Hoyos 2000 supported this model
o Used transgenic mice with altered Lck activity
o Showed that thymocytes carrying an MHC class II restricted TCR develop into functional CD8+ T cell when Lck activity is reduced (inactive Lck transgene redirect MHC class II–restricted thymocytes to the CD8 lineage)
o Conversely thymocytes carrying class I restricted TCR develop into functional CD4 T cells when Lck activity is increased (constitutively active Lck transgenes can redirect MHC class I–restricted thymocytes to the CD4 lineage)
o These quantitative differences in Lck signal control CD4/CD8 lineage decision
anergy study
Liu et al (2002) = DCs induce tolerance in T cells with avidity for self, in absence of inflammation
• Injected mice with dying syngeneic TAP-/- splenocytes loaded with small amounts of ovalbumin
- TAP KO means cells cannot directly present OVA presentation of dying cells limited to recipient DCs in situ
• Splenocyte injection led to efficient antigen presentation on MHC class I of recipient DCs BUT failed to induce maturation of DCs (no alterations in surface markers associated with terminal differentiation (e.g. MHC class II, CD40, CD80/86)) = no change in MFI
• Induced potent CD8+ T cell proliferation BUT no changes in surface markers (e.g. upregulation of IL-2R, downregulation of LN homing receptor) or cytokine production
• T cells progressively deleted = number of T cells in spleen, lymph node and blood decreased drastically by day 10
• Shows that DC antigen presentation leads to CD8+ T cell anergy in steady state
when splenocytes injected in combination with CD40 (induced DC activation) –> T cells activated, IFNy and IL-2 secretion
• Focused exclusively on tolerization of CD8+ cells = unclear whether same mechanisms lead to toleration of CD4+ T cells too
IL-10 important for Treg
Asseman et al (1999) = IL-10 is essential for Tregs that inhibit intestinal inflammation
• Previous studies showed that Th1-mediated colitis could be induced in SCID mice by transfer of CD4+ CD45RBhigh T cells and prevented by cotransfer of CD45RBlow cells = contains population of Tregs capable of controlling intestinal inflammation
• Study found that transferring CD45RBlow cells from IL10 KO mice could not rescue colitis
- Colonic inflammation was similar in mice transferred with just CD4+ CD45RBhigh T cells, and those transferred with CD4+ CD45RBhigh T cells and IL10 KO CD45RBlow cells = histology revealed high inflammatory cell infiltrate, marked epithelial hyperplasia, depletion of goblet cells, cytofluorography revealed accumulation of IFNy and TNFa secreting T cells
• Treatment of mice transplanted with WT CD45RBlow cells with anti-IL10R also ablated protection from colitis
• This said, it cannot be assumed that a model that depends on cell expansion in a lymphopenic host, of another species, mimics human IBD
• Roles of cytokines are not always conserved between mice and humans
FOXP3 evidence
Hori et al (2003)
- Showed that FOXP3 is expressed in CD4+ Treg cells (RT-PCR), and that retroviral gene transfer of FOXP3 cDNA converts naïve T cells towards a regulatory T cell phenotype
CLINICAL: IPEX
• Autoimmune condition caused by mutation in FOXP3 gene necessary for Treg development and function
• Severe multiorgan disease = enteropathy (diarrhoea, vomiting, colitis, gastritis), T1D, dermatitis, alopecia, thyroid and renal dysfunction, arthritis……
• Requires treatment with BMT in early childhood
FOXP3 mutations have also been found to account for the immunopathological phenotype seen in Scurfy mouse mutants, who display an absence of Treg cells, and develop a CD4+ T cell-mediated lymphoproliferative disease that results in multiorgan lymphocyte infiltration and premature death. Evidence for this was obtained by Brunkow et al (2001), who used genetic sequencing to demonstrate that the Scurfy phenotype was due to a frameshift mutation in FOXP3, leading to the production of a truncated scurfin protein that lacked the fork-head domain required for interaction with DNA, and hence transcriptional regulation. They also showed that genetic complementation of the entire FOXP3 gene into the Scurfy mouse oocytes was sufficient to restore normal tolerance, with the adult mice showing no signs of autoimmune disease, and possessing normal livers, spleens and lymph nodes, illustrating that FOXP3 is both necessary and sufficient for normal self vs non-self discrimination.
negative selection = first evidence
EXP: Kappler et al 1987 – first evidence for negative selection
o It was known that T cells expressing a TCR containing the Vβ domain Vβ17a reacted strongly with the MHC II protein IE.
o Mice were engineered to express the protein IE and a Vβ17a-specific fluorescent antibody was used to label the TCRs and monitor T-cell fate.
o In transgenic mice, the number of Vβ17a+ T cells in the thymus was high among immature thymocytes but low among mature Treg or Tconv, in contrast to WT mice which had high numbers of mature Vβ17a+ T-cells in the thymus and periphery.
o This suggests that clonal deletion of IE specific cells was occurring as a result of a high TCR avidity for the self-peptide MHC complex
o Numbers of VB17a T cells in periphery varied between different IE+ mice strains
- May be due to differences in VB17a+ repertoire selected in thymus by different MHC haplotypes, or differences in dose of IE expressed in different animals
- Should have repeated experiments in mice homozygous for same MHC haplotype
AIRE evidence
EXP: Anderson et al 2002
• Used Cre/lox site specific recombination techniques to create AIRE -/- mice
• Observed lymphocyte infiltrates autoimmune pathologies in multiple organs
• To establish specific function of AIRE expression in the thymus he used PCR observed greater level of AIRE expression in the thymus than any other major peripheral organ
• AIRE deficient mice showed a specific reduction in ectopic expression of genes encoding peripheral antigens (sqRT-PCR)
• Performed thymus graft from AIRE-/- or WT littermate into an athymic WT recipient + observed significant levels of autoantibodies in the sera of 3 mice with AIRE deficient thymus but none in the 6 WT hosts
• Suggested thymic AIRE translation necessary and sufficient for T cell central tolerance
importance of DCs in self-tolerance
EXP: Ohnmacht et al (2009) = importance of DCs in self-tolerance
• Used Cre-lox to selectively ablate DCs and Langerhans cells from mice
- Crossed CD11c-Cre mice with mice expressing diphtheria toxin under control of loxP-flanked neomycin resistance cassette from ROSA26 locus
• Mice showed increased frequencies of CD4+ SP thymocytes (as measured by flow cytometry), infiltration of CD4 T cells into peripheral tissues, severe spontaneous autoimmunity (e.g. reduced body weight + survival, inflammatory bowel disease, neutrophilia, splenomegaly)
- But no change in CD8 SP cells = indicates DCs are less important in negative selection of these cells
• Reconstituted WT mice with bone marrow from DC-depleted mice = developed pathology
• Mixed BM chimeras receiving BM from DC-depleted and WT mice remained healthy
• Relative contribution of DCs and mTEC to negative selection is disputed DC transplantation studies suggest that DCs are primary mediators of negative selection for tissue-restricted antigens rather than mTEC
evidence that Treg selection occurs at higher avidity
Lee et al (2012) = crucial evidence that higher TCR avidity favours Treg selection over Tconv selection
• Used well characterised model of tissue-specific antigen = RIP-mOVA line, in which rat insulin promoter drives expression of membrane-bound ovalbumin incubated with T cells expressing TCRs with varying affinity for OVA peptide assessed developmental effects through flow cytometry (detecting proportion of cells w FOXP3 and CD25 expression)
• They showed that anti-OVA T-cells from transgenic mice expressing low-reactivity TCR were selected to the Tconv lineage
• By contrast, T-cells with TCR of higher reactivity predominantly developed as Treg
• And anti-OVA T-cells expressing the highest affinity TCR were either negatively-selected or became Treg
• This provides strong evidence for the affinity model of lineage selection
• Data needs to be validated in other model systems, with other antigens
insights in Treg selection
Lio et al (2008) = new insights into Treg selection
• CD25highFOXP3-CD4+CD8- cells previously suggested as Treg precursor population
• Performed intrathymic transfer with various thymic subsets assessed frequency of cells that upregulated FOXP3 expression to determine whether subsets were enriched for Treg precursors
• Much greater proportion of CD4SP CD25high cells acquired FOXP3 expression than CD25low (FC) = confirmed this is a more Treg-enriched population
• Then studied signals required for Treg selection
- Proportion of intrathymically transferred CD25highFOXP3-CD4+CD8- cells that differentiated to Treg cells was the same in WT and MHCII KO mice non-TCR signal likely sufficient to induce selection to Treg lineage at this stage
- In contrast, small frequency of CD25lo cells that became FOXP3+ in WT were ablated in MHC KO host supports theory that CD25lo thymocytes are at earlier stage in Treg cell development, do require TCR-derived signals
- Further experiments showed that IL-2 signalling sufficient to induce Treg selection in CD25highFOXP3-CD4+CD8 but not CD25lo two-step process of TCR signalling and cytokine signalling required for Treg selection
