Selection of T‐cell receptors with a recurrent CDR3β peptide‐contact motif within the repertoire of alloreactive CD8<sup>+</sup> T cells

Scifo, Caroline; Mekaelian, Laura; Munyazesa, Elisaphane; Schmitt-Verhulst, Anne-Marie; Guimezanes, Annick

doi:10.1002/eji.201141494

“…Of the 100 K b -binding peptides selected for tetramer screening, 17 were recognised by >5% of the enriched alloreactive T cell population. INFDFNTI, previously reported to be recognised by a large proportion of the polyclonal alloreactive T cell population of B10.BR mice (25) was bound by an average of 3.7% of male B10.BR cells. To examine factors which might contribute to the immunogenicity of different pMHC, we used spectral intensity as an approximation for relative peptide abundance and IC50 to predict binding affinity of the peptide for H-2K b .…”

Section: Discussionmentioning

confidence: 86%

“…96 peptides were drawn from the common peptide pool, and their identity was confirmed by a direct comparison between the mass spectra obtained from synthetic and eluted natural peptides (Supplementary Figure 8). A further four peptides had been previously identified as alloreactive CD8 + T cell epitopes in B10.BR mice (11,25). Three of these four epitopes were detected within the common pool.…”

Section: Profiling the Tissue-specific Immunopeptidomes Of Hepatocytes Skin And Spleenmentioning

confidence: 99%

The self-peptide repertoire plays a critical role in transplant tolerance induction

Son

¹

,

Faridi

²

,

Paul-Heng

³

et al. 2021

Journal of Clinical Investigation

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While direct allorecognition underpins both solid organ allograft rejection and tolerance induction, the specific molecular targets of most directly-alloreactive CD8 + T cells have not been defined. In this study, we used a combination of genetically-engineered MHC class I (MHC I) constructs, mice with a hepatocyte-specific mutation in the class I antigen-presentation pathway and immunopeptidomic analysis to provide definitive evidence for the contribution of the peptide cargo of allogeneic MHC I molecules to transplant tolerance induction. We established a systematic approach for the discovery of directly-recognised pMHC epitopes, and identified 17 strongly immunogenic H-2K b -associated peptides recognised by CD8 + T cells from B10.BR (H-2 k ) mice, 13 of which were also recognised by BALB/c (H-2 d ) mice. As few as five different tetramers used together were able to identify a high proportion of alloreactive T cells within a polyclonal population, suggesting that there are immunodominant allogeneic MHC-peptide complexes that can account for a large component of the alloresponse. 5 Results Single chain trimer constructs exclude presentation of endogenous peptides.In preceding studies(1, 3), we have used AAV vectors encoding the donor MHC I HC. Within transduced hepatocytes, allogeneic HC associates with native b2m and the resulting heterodimers are loaded with a repertoire of endogenous peptides (Figure 1A). To express allogeneic MHC I at high levels on recipient hepatocytes while excluding the presentation of naturally processed peptides, we engineered SCT constructs, each encoding the HC of H-2K b , b2m and a single, defined H-2K b -restricted peptide [SIINFEKL (SIIN) or KIITYRNL (KIIT), Figure 1A], and packaged them in hepatocyte-specific AAV2/8 vectors.Sequences are shown in Supplementary Figure 1. Transgene expression in hepatocytes was close to maximal by d7 following intravenous (iv) inoculation, and persisted through to at least d100, no significant increases in serum aspartate aminotransferase (AST) or alanine aminotransferase (ALT) levels were observed, and minimal cellular infiltration was detected by histology (Supplementary Figure 2). SCT molecules were expressed on transduced hepatocytes at equivalent levels to the heterotrimer formed by transgenic H-2K b HC with native b2m and peptide (Figure 1B). To demonstrate exclusion of naturally processed peptides, we co-transduced B10.BR (H-2 k ) hepatocytes with AAV vectors encoding full-length chicken ovalbumin (OVA) and either HC-K b or SCT-K b -KIIT, and stained them with a monoclonal antibody, 25D-1.16, which is specific for the OVA peptide SIINFEKL complexed with K b . K b -SIINFEKL was only detected at the surface of cells co-transduced with HC-K b and not those expressing SCT-K b -KIIT (Figure 1C-D). We extended this analysis to the broader endogenous peptide repertoire of K b -transduced hepatocytes using immunoaffinity purification with the H-2K b -specific antibody K9-178, followed by reverse phase high performance liquid chromatography (RP-HPLC...

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“…Of the 100 K b -binding peptides selected for tetramer screening, 17 were recognised by >5% of the enriched alloreactive T cell population. INFDFNTI, previously reported to be recognised by a large proportion of the polyclonal alloreactive T cell population of B10.BR mice 25 was bound by an average of 3.7% of male B10.BR cells.…”

Section: Discussionmentioning

confidence: 86%

“…For all peptides, p < 0.05. A further four peptides had been previously identified as alloreactive CD8 + T cell epitopes in B10.BR mice 11, 25 . Three of these four epitopes were detected within the common pool.…”

Section: Resultsmentioning

confidence: 99%

The self-peptide repertoire plays a critical role in transplant tolerance induction

Son

¹

,

Faridi

²

,

Paul-Heng

³

et al. 2020

Preprint

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While direct allorecognition underpins both solid organ allograft rejection and tolerance induction, the specific molecular targets of most directly-alloreactive CD8+ T cells have not been defined. In this study, we used a combination of genetically-engineered MHC I constructs, mice with a hepatocyte-specific mutation in the class I antigen-presentation pathway and immunopeptidomic analysis to provide definitive evidence for the contribution of the peptide cargo of allogeneic MHC I molecules to transplant tolerance induction. We established a systematic approach for the discovery of directly-recognised pMHC epitopes, and identified 17 strongly immunogenic H-2Kb-associated peptides recognised by CD8+ T cells from B10.BR (H-2k) mice, 13 of which were also recognised by BALB/c (H-2d) mice. As few as five different tetramers used together were able to identify almost 40% of alloreactive T cells within a polyclonal population. To our knowledge, this represents the first example of such an approach in the context of direct allorecognition.

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“…Ninety-six peptides were drawn from the common peptide pool, and their identity was confirmed by a direct comparison between the mass spectra obtained from synthetic and eluted natural peptides (Supplemental Figure 8). An additional 4 peptides had been previously identified as alloreactive CD8 + T cell epitopes in B10.BR mice (11,25). Three of these 4 epitopes were detected within the common…”

Section: Resultsmentioning

confidence: 99%

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Selection of T‐cell receptors with a recurrent CDR3β peptide‐contact motif within the repertoire of alloreactive CD8⁺ T cells

Cited by 5 publications

References 41 publications

The self-peptide repertoire plays a critical role in transplant tolerance induction

The self-peptide repertoire plays a critical role in transplant tolerance induction

The self-peptide repertoire plays a critical role in transplant tolerance induction

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Selection of T‐cell receptors with a recurrent CDR3β peptide‐contact motif within the repertoire of alloreactive CD8+ T cells

Cited by 5 publications

References 41 publications

The self-peptide repertoire plays a critical role in transplant tolerance induction

The self-peptide repertoire plays a critical role in transplant tolerance induction

The self-peptide repertoire plays a critical role in transplant tolerance induction

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Selection of T‐cell receptors with a recurrent CDR3β peptide‐contact motif within the repertoire of alloreactive CD8⁺ T cells