A detailed assessment of the TCR repertoires of antigen-specific T cells is a prerequisite for a better understanding of human anti-viral immunity. Here we systematically examined the α-TCR repertoires of memory CD8 T cells reactive against the influenza A viral epitope, M158-66, restricted by HLA-A2.1. The M158-66-specific, clonally diverse VB19 CD8 T cells expressed α-chains from several VA families with different CDR3 sizes. A unique feature of these α-TCRs was the presence of poly-Gly/Ala runs in the CDR3, fitting to an AGA(Gn)GG-like amino acid motif. These non-template-encoded poly-Gly/Ala runs in the CDR3 of the M158-66-specific memory pool were significantly enriched over that in naïve thymocytes, indicating that Gly/Ala runs provided a selective advantage in antigen-driven repertoire development in the periphery. These poly-Gly/Ala runs in the CDR3 of α- and β-chains might provide enhanced TCR flexibility during antigen recognition.
The mechanisms that shape T cell memory through α-TCR selection have been difficult to delineate due to the technical restraints associated with the lack of VA-family specific mAbs and T cell ability to co-express two α-chains (31
). Nevertheless, our molecular cloning techniques demonstrate that the influenza A M158-66
-specific T cell memory contains a number of additional features contributed by α-TCR diversity. These TCR α-chains that paired with the VB19 β-chains were of eleven VA families with three remarkably different sizes in CDR3α (). Given that the M1-specific clonotypes from different VA families express different CDR1α and CDR2α (), proper accommodation of different CDR1α and CDR2α to the M158-66
-:HLA-A2 might occur if the CDR3α could undergo conformational adjustment. In this regard, enrichment of Gly and Ala might provide increased structural flexibility in CDR3α and satisfy this criterion.
It is commonly accepted that the fine specificity of epitope recognition is due to structural complementarity of CDR3α and CDR3β to MHC-presented immunogenic peptides, under conditions where CDR1 and CDR2 orient the TCR α- and β-chains to MHC molecules. In contrast to antibodies that usually have large surfaces with complementarity to their cognate antigens (35
), only 21-34% of the αβ-TCR's surfaces are in direct contact with pMHC complexes (16
). Moreover, the contribution of CDR3α and CDR3β is relatively small, representing 21% and 24% on average of Vα and Vβ domains, respectively. These properties of TCR-pMHC interactions impose strict requirements on α- and β-chains. For example, side chains of amino acids located within the CDR3 must have optimized sizes and charges to interact with the foreign peptide, and CDR3 of α- and β-chains ought to have the similar sizes (25
). However, we show examples where short CDR3α (12 amino acids) pair with one amino acid longer VB19-CDR3β (counting from C (CAS) to F (FGXG)), similar to crystallized αβ-TCR expressed by M158-66
-specific clone JM22 (37
). The CD8 T cells from VA27 and VA8 families in five studied individuals mostly expressed these short CDR3α loops. In contrast, longer CDR3α sequences (14 and 15 aa) were found with VA families other than VA27 and VA8. These “non-VA27” VB19+
T cells share α-JA42 chains with VA27 T cells (), but express different CDR1 and CDR2 of their α-chains (). Given that VB19 cells express rigid CDR3β fitting into the “
IRSS” or “IGS”-like motifs, a plausible explanation that structurally different VA-domains are used to recognize influenza epitope is that the CDR3α loops undergo significant conformation (38
) associated with poly-Gly/Ala runs. We therefore suggest that the CDR3α bearing long poly-Gly/Ala strings allow CDR1α and CDR2α encoded by different VA families to be used during influenza antigen recognition, and we discuss the theoretical basis for this suggestion below.
-gene encoded products are not unique in the sense of containing two Gly runs, since 7 out of 51 AJ
genes encode two, and even three, Gly. However, 59% (37/63) of the clonotypes with CDR3α with 15 aa belong to this particular JA42 family (). It seems that the combination of long CDR3α with poly-Gly/Ala runs provides flexibility for αβ-TCR to bind to the M158-66
:HLA-A2 epitope, since we defined only eight clonotypes from “non-VA27” families (namely, VA10, 8.6, VA34 and VA35) that expressed short CDR3α of 12 amino acids (). Gly is a unique amino acid because it lacks a side chain, and Ala contains only a methyl group as a side chain. It has been shown that proteins whose functions depend on adjustment to ligands often contain flexible loops. Usually Gly is located within these loops, providing flexibility in protein-protein or protein-ligand interactions. For example, poly-Gly strings have been found in the HIV protease flap region, in β1,4-galactosyltransferase-I, fructose-1,6-bisphosphate aldolase and other enzymes (39
) . TCR contact with the pep:MHC molecule also follows the same rule. Thermodynamic studies of three TCR-pMHC binding, including αβ-TCR from M158-66
-specific clone JM22, revealed that this process correlates with considerable conformational adjustment in CDR3α and CDR3β (42
). For instance, Malissen and co-workers reported that KB5-C20 (TCR specific to pKB1/H-2Kb
) exhibits large conformational alteration in the CDR3β (6 amino acids longer than CDR3α) for proper accommodation to pMHC (15
). Recently, the same group reported that similar structural flexibility might be observed in CDR3α (BM3.3 specific to two peptides with low sequence similarity presented by H-2Kb
). Based on these studies, the authors concluded that αβ-TCR propensity to modify its complementarity surface, mostly in the CDR3, might be the origin of αβ-TCR intrinsic ability to interact with the different epitopes. In line with these studies is the observation of structural flexibility of the αβ-TCRs expressed by human T cell clones reactive against Tax11-19
peptide (from HTLV) presented by HLA-A2.1(44
). Remarkably, these clones expressed β-TCRs (VB13.1) that contained a PG×G motif in the CDR3β and efficiently recognized the original epitope and its variants. Another proof outlining the importance of TCR structural flexibility in epitope recognition comes from the crystallization of the αβ-TCRS expressed by clone LC13 specific to EBNA-3339-347
peptide presented by HLA-B8. In this case, AlaGlyGly runs were contained in the CDR3β (46
The occurrence of poly-Gly runs in β-TCRs with a long CDR3 is attributed to the D region where VDJ
-gene transcription in three “open-reading-frames” would encode multiple Gly. This rule, however, cannot be applied to α-TCRs lacking D-encoded regions. The existence of VB19 clones specific to flu-M158-66
that utilize α-TCRs from different VA families provide an interesting example of epitope recognition where germ-line encoded segments of α-chains (i.e. SQG from AJA42
gene) contact M158-66
, while segments created by AV/AJ42 recombination are positioned outside M158-66
:HLA-A2 and could be flexible due to Gly/Ala enrichment (37
). A recent study of the αβ-TCR (from clone JM22 specific to M158-66
-epitope) before and after binding to M158-66
:HLA-A2 revealed that the CDR3α loop swiveled and made about 5Å outward shift (37
). It should be mentioned that CDR3α loop from JM22 contains only 12 amino acids. Therefore, we feel confident that the CDR3α with 14 and 15 aa defined in our study might have considerably more rotation and movement during M158-66
:HLA-A2 binding and allow the CDR1α and CDR2α from different VA families to interact with HLA-A2 α1-helix.
A less explored field in human immunology is the analysis of the molecular nature of the pre-immune α-TCR repertoire. In our study, we could not exclude the possibility that poly-Gly/Ala runs might be a result of preferential AV27/AJ42 recombination where long CDR3α might have increased frequencies of Gly and Ala. If this were the case, we would have expected to see increased observations of Gly and Ala in CD4−8+ and CD4+8− thymocytes regardless of class I and II HLA-restrictions. It is important to point out that we examined the transcriptional profiles of α-TCRs where VA27/JA42 transcripts might encode functional (restricted by HLA-A2.1) and non-functional TCR α-chains. Following extensive sequencing analysis of AV27/AJ42
gene recombination and considering CDR3α sizes, we concluded that Gly and Ala have similar frequencies with other amino acids encoded by non-template segments of the VA27/JA42 rearranged genes ( and Supplemental, Table II
). Although two Gly are derived from AJ42
-gene as was expected since we examined VA27/JA42 transcripts, the non-template segments of long CDR3α (i.e. 14 and 15 aa) were not Gly- and Ala-enriched. Therefore, we conclude that selection for poly-Gly/Ala runs was driven in response to M158-66
epitope during influenza exposure rather than by a gene recombination.
The flexibility of αβ-TCR structure might be an important factor in the fate of memory T cells. In the case of influenza M158-66
-specific cells, the poly-Gly/Ala runs do not contact M158-66
or HLA-A2 directly, based on the crystallization of the representative M158-66
-specific JM22 clonal αβ-TCR and its variants (37
). Although the recognition M158-66
:HLA-A2 is a function of VB19 β-chain (~70% of interactive interface), the α-TCRs with a highly flexible CDR3 might be used to recognize structurally different antigens, thus contributing to the pattern of T cell cross-reactivity which we have observed in “heterologous immunity” (47
). If this is the case, influenza-specific VB19 T cells with IRSS in CDR3β might engage CDR1α and CDR2α during recognition of other p:HLA-A2, perhaps further enhancing the immunodominance of VB19 T cell clones.
Here we propose a three-step model explaining TCR interaction with the M158-66/HLA-A2.1 complex (). In the first step, CDR1β and CDR2β (β-VB19) contact the α2-helix of HLA-A2.1, pivoting the CDR3β to the peptide wherein R98 anchors the β-chains to HLA-A2 and S99 interacts with the M158-66 peptide. In the second step, the SQG (AJ42-gene encoded) anchors the CDR3α loop to Gly61 (M158-66), and the long CDR3α (poly-Gly/Ala) undergoes conformational change. Since AGA(G/A)G in CDR3α imposes minimal energy requirement to change shape, this leads to the third step, where engagement of different CDR1,2α (from any of VA-domain) is sufficient for final TCR:M158-66/HLA-A2.1 docking. The key element of this model is a long poly-Gly/Ala moiety, which allows CDR3 to be extremely flexible and adjust different TCR V-domains to the same pMHC complex. If long CDR3α- and/or CDR3β are more flexible and accommodate αβ-TCRs to different pMHC shapes and charges, then cross-reactivity against different antigens might be a major factor in memory formation. It is noteworthy mentioning that in our studies we also defined, based on tetramer binding, “non-VB19” cells that were able to recognize M158-66/HLA–A2 epitope. Remarkably, they also expressed long, 15 aa, CDR3β with a GXGG motif (Naumov, unpublished).
Proposed model of poly-Gly/Ala insertion in CDR3α loop
The early studies of the cytotoxic CD8 T cell lines and clones reactive against M157-68
-expressing targets cells revealed that M157-68
peptide modifications in positions 58-60 were well-tolerated, while modifications in positions 61-65 could abrogate CTL response. In the last case, however, diminished cytotoxicity was not absolute and depended on amino acid substitutions. Interestingly, the T cell clones derived presumably from VB19 family have different patterns of epitope dependency in the CTL assay (10
). Our own study demonstrated that small populations of the VB19 T cells are able to proliferate and to produce IFN-α in response to influenza M158-66
). Although we did not define yet the structure of the cross-reactive αβ-TCRs, these observations suggest that conformational flexibility of αβ-TCRs and clonal diversity of reactive T cells might be the best way to cope with different antigens.
In conclusion, we would like to suggest that the immune response evolves in a way where it engages T cells with structurally different αβ-TCRs specific to cognate antigens leading to an intrinsic capacity of these T cells to interact with different p:MHC shapes and charges. It is tempting to speculate that presence of multiple memory CD8 T cell clones of diverse specificities due to adjustable antigen receptors is the best way to optimize immune memory to ever-changing antigenic environment.