Increased Activity in the T Cell Effector Phase and Enhanced T Cell Repertoire Target-Tissue Stability Distinguish Alloreactivity Across Major Versus Minor Histocompatibility Barriers

Graft-versus-host disease (GVHD) occurs when transplanted donors’ T cells recognized the recipients’ antigens and damaged host tissues and cells, particularly the skin, gut and liver in the acute setting.  Although it is well known GVHD is more aggressive and manifests more quickly across major versus minor histocompatibility barrier, little is known comparatively about the donor T cell activation and T cell repertoire changes.

To investigate temporal and spatial events of GHVD development, side-by-side transplants were conducted into major and minor-mismatched murine recipients (Balb.c and Balb.b) using hematopoietic cells from the same donor strain(B6). In both models, T cells home to nodal sites by day 3, proliferate, and exit to GVHD target tissues by day 6. Additionally, expression of homing and activation markers was equivalent for all markers examined on day 3. However, tissue migration and proliferation were reduced in the minor model. By day 6, minor-mismatched T cells had increased CD62L retention and reduced P-selectin and CD44 expression.  We also found fewer MHC-matched T cells producing IFN-g and TNF-a.

Our data show that early events of donor T cell activation are similar in both models, suggesting that the delayed onset and attenuated disease GVHD seen across minor barriers arise from temporal differences in the effector phase, rather than the initiation phase, of GVHD.

To further understand the differences across major versus minor histocompatibility barriers on the T cell repertoire and patterns of T cell alloreactivity, we collected a sample of T cells from donor mice used for transplantation, and also sampled gut tissues from syngeneic, major and minor- mismatched transplanted mice on day 9, 9 and 30 respectively at times when the allogeneic groups of mice showed severe GVHD symptoms.  

To reduce the background of high percentage of TCR pseudogenes in mouse genome, 5’RACE starting from RNA samples and deep sequencing of TCRa and TCRb were applied to investigate whether TCR repertoire of the major and minor-mismatched mice were skewed with clonal expansion, and how among the major and minor-mismatched mice. While we hypothesized that the major MHC mismatched group would have lower diversity because of expanded clones associated with the GVHD, the shannon index of TCRa indicated gut TCR repertoire of major and minor mismatched mice have greater diversity than syngeneic group(P<0.05). We did not see differences in the shannon index of diversity on examination of the TCRb repertoire.

Contrary to our expectation, that the TCR repertoire of major mismatch would be skewed towards representation of a few highly expanded clones in the gut, we in fact saw that the TCR repertoire in these mice appeared less skewed. The TCR repertoire of the gut was more highly related among individuals in the  major mismatch groups than among or between the other groups. This strongly suggests a more reproducible repertoire structure.   

To examine if certain T cell clones were more likely to appear reproducibly in the major and/or minor mismatch setting, we compared the shared clones across the major-mismatched transplanted mice.  Bhattacharyya coefficients showed that the major-mismatched mice shared more clones with the minor-mismatched group than the syngeneic groups(P<0.05).  A total of eight shared TCRa clonotypes among major-mismatched mice are also detected. The common CDR3 clonotypes might be associated with GVHD. We found 7 of them are also in donor CD4 memory cells’ repertoire; and one is present in both donor’s CD4 and CD8 memory cells. One of the complementarity determining regions sequences is “AASYQGGRALI”  which is also in common among minor-mismatched mice.

We also measured the repertoire similarity between donor T cells and the gut TCR repertoires of three groups. Bhattacharyya coefficients of TCRa and TCRb between donor T cells and major-mismatched gut repertoire is greater than donor T cells with either syngeneic or minor-mismatched repertoire(P<0.05), which suggest that the major-mismatched mice has more T cells homing to gut which might be associated with GVHD.  

Our sequencing data showed that major and minor-mismatched transplantation might not cause the clonal expansion in the gut TCR repertoire, but their repertoire patterns are different from the syngeneic groups, which might be associated with T cell alloreactivity and GVHD.