CD4 T cell responses against chromogranin A peptide: a critical interaction for disease onset, now defined at level of crystal structure
by Adam Burrack, PhD
Broad immune suppression or T cell depletion studies are not broadly applicable solutions to prevent type 1 diabetes (T1D) onset or rejection of transplanted pancreatic islets. These treatments would leave patients too vulnerable to infections or cancer. The most specific way – and therefore the most likely clinical approach – to try to prevent beta cell destruction by T cells of the immune system – in either disease onset or islet transplant scenarios – is to figure out the specific protein/peptide target of the T cells, and to prevent that response. Easier said than done. Two recent papers both from the Barbara Davis Center in Denver and investigating a key T cell response shed more light on the mechanisms underlying autoimmunity against beta cells.
In an article in the “cutting edge” section of the Journal of Immunology, Dr. Kathryn Haskins’ research group published a paper showing that NOD mice genetically engineered to not express the chromogranin A molecule – which is present within the secretory granules with insulin, and is a key target of autoreactive T cells in both humans with T1D and in the mouse model of T1D – do NOT develop diabetes.
This definitive proof that ChgA is a critical target very early in the diabetic disease process has been a long time coming. As we previously described Dr. Haskins isolated a number of T cell lines from NOD mice that transferred diabetes in the late 1980s. These T cell lines have been used since then to study T cell biology and beta cell destruction. In 2010, Dr Haskins and her post-doctoral researcher Thomas Delong – a recent ADA Pathway recipient – published a paper demonstrating a peptide derived from chromogranin A is the target of the best-studied of these T cell lines, the BDC2.5. They went on to show that chromogranin A is a target of autoreactive T cells in humans with T1D as well. What had been less clear is the importance of ChgA in the onset of T1D – in other words is this response a cause of disease or a consequence of disease?
The chromogranin A-deficient NOD mouse was designed to test this question. In short, ChgA-deficient NOD mice do not develop type 1 diabetes, and have greatly reduced T cell infiltration into their pancreas. In these ChgA-deficient mice, lead author Rocky Baker could still find CD4 T cells which bound the MHC tetramer reagent designed to find ChgA-specific T cells, but phenotyping by flow cytometry indicated that these potentially pathogenic T cells remained ‘naïve’, rather than activated, and did not leave the spleen to traffic into the pancreas. As an immunologist this makes sense: if the target peptide of these T cells was not present in the pancreas, the T cells would have had no target to search and destroy. So, they remained naïve. As a side-note, it is interesting that these cells still developed in the thymus and made it to the spleen. Interestingly, the ChgA-deficient NOD mice still developed inflammation in their salivary glands, which is another site of autoimmune T cell attack in NOD mice, suggesting that ChgA is not a key peptide precipitating autoimmunity against the salivary glands.
These results suggest that ChgA-specific T cells develop in NOD mice lacking the ChgA peptide in the pancreas, but in the absence of their target in the pancreas these T cells do not enter the pancreas or participate in disease onset. The key take-home of this paper is that without this specific CD4 T cell response, NOD mice do not develop diabetes – suggesting that the CD4 T cell response to ChgA is as critical as the anti-insulin response: to the best of my knowledge, these are the only two T cell responses whose absence prevents diabetes onset. Therefore, studies investigating methods to inhibit or remove these T cells from NOD mice take on a renewed urgency: if this T cell response can be prevented, perhaps epitope spreading would be prevented and disease would not occur in at-risk animals.
There was another exciting development related to T1D development and the role of the chromogranin A peptide. John Kappler’s research group – former interim Research Director at the BDC and recent winner of the Wolf Prize in Medicine, has identified the ‘binding register’ of the chromogranin A peptide in the major histocompatibility complex molecule of the NOD mouse!
This is important for at least two reasons. First, the theoretical crystal structure of the ChgA peptide bound to the MHC molecule of the NOD mouse (the study of crystal structures of proteins bound to MHC is a field unto itself, in which John Kappler is one of a handful of world-renowned experts) showed an “open pocket” at the beginning of the peptide-binding groove. This is very abnormal. For a peptide to bind the MHC molecule it had been thought that peptide needs to both (1) “fill the groove”, and (2) have permissive amino acids in particular “docking residues” for the MHC molecule. For this paper Dr. Kappler’s group solved the crystal structure of the ChgA-derived peptide loaded in the MHC molecule of the NOD mouse. The known ChgA peptide fulfilled the second criteria, but not the first. Dr. Kappler shows in this ground-breaking paper that a sequence of 4 amino acids from a different section of the ChgA protein fill the peptide 1-4 pocket in the MHC molecule in this crystal structure. This means that Dr. Delong had the peptide correct and the anchor residues correct, but pocket 1-4 is not left unoccupied, it is in fact occupied by a different chunk of the ChgA peptide. Second, this concept, that a different chunk of a protein can fill the open portion of the MHC groove in the presence of a good-binding peptide in the anchor residues, is a new one for the field of X-ray crystallography.
To translate this to English, now we know how ChgA binds the MHC molecule. This could facilitate the development of better tools for researchers to use to find these T cells – including opening minds to this new possibility of peptide-MHC binding – and to track the behavior these T cells over disease course and in the presence of therapies intended to stop disease onset or promote islet transplant tolerance. Very exciting news, indeed!