The durable alloantibody responses that develop in organ transplant patients indicate long-lived plasma cell output from T-dependent germinal centres (GCs), but which of the two pathways of CD4 T cell allorecognition are responsible for generating allospecific T follicular helper (TFH) cells remains unclear. This was addressed by reconstituting T-cell deficient mice with monoclonal populations of TCR-transgenic CD4 T cells that recognised alloantigen only as conformationally-intact protein (direct pathway) or only as self-restricted allopeptide (indirect pathway), and then assessing the alloantibody response to a heart graft. Recipients reconstituted with indirect-pathway CD4 T cells developed long-lasting IgG alloantibody responses, with splenic GCs and allospecific bone marrow plasma cells readily detectable 50 days after heart transplantation. Differentiation of the transferred CD4 T cells into TFH cells was confirmed by follicular localisation and by acquisition of signature phenotype. In contrast, IgG alloantibody was not detectable in recipient mice reconstituted with direct-pathway CD4 T cells. Neither prolongation of the response by preventing NK cell killing of donor dendritic cells, nor prior immunisation to develop CD4 T cell memory altered the inability of the direct-pathway to provide allospecific B cell help. CD4 T cell help for GC alloantibody responses is provided exclusively via the indirect-allorecognition pathway.

SummaryThe differentiating bacterium Caulobacter crescentus produces two different cell types at each cell division, a motile swarmer cell and an adhesive stalked cell. The stalked cell harbours a stalk, a thin cylindrical extension of the cell surface. The tip of the stalk is decorated with a holdfast, an adhesive organelle composed at least in part of polysaccharides. The synthesis of the stalk and holdfast occur at the same pole during swarmer cell differentiation. Mutations in the hfaABDC gene cluster had been shown to disrupt the attachment of the holdfast to the tip of the stalk, but the role of individual genes was unknown. We used lacZ fusions of various DNA fragments from the hfaABDC region to show that these genes form an operon. In order to analyse the relative contribution of the different genes to holdfast attachment, mutations were constructed for each gene. hfaC was not required for holdfast attachment or binding to surfaces. The hfaA and hfaD mutants shed some holdfast material into the surrounding medium and were partially deficient in binding to surfaces. Unlike hfaA and hfaB mutants, hfaD mutants were still able to form rosettes efficiently. Cells with insertions in hfaB were unable to bind to surfaces, and lectin binding studies indicated that the hfaB mutants had the strongest holdfast shedding phenotype. We determined that HfaB and HfaD are membrane-associated proteins and that HfaB is a lipoprotein. Purification of stalks and cell bodies indicated that both HfaB and HfaD are enriched in the stalk as compared to the cell body. These results suggest that HfaB and HfaD, and probably HfaA, serve to anchor the holdfast to the tip of the stalk.

Essential help for long-lived alloantibody responses is theoretically provided only by CD4 T cells that recognise target alloantigen, processed and presented by the allospecific B cell. We demonstrate that in an alloresponse to multiple MHC disparities, cognate help for class-switched alloantibody may also be provided by CD4 T cells specific for a second ‘helper’ alloantigen. This response was much shorter-lived than when help was provided conventionally, by helper T cell recognition of target alloantigen. Nevertheless, long-lasting humoral alloimmunity developed when T cell memory against the helper alloantigen was first generated. Co-stimulatory blockade abrogated alloantibody produced through naive helper T cell recognition of target alloantigen, but crucially, blockade was ineffective when help was provided by memory responses to the accessory helper alloantigen. These results suggest that memory helper T cell responses against previously-encountered graft alloantigen may be the dominant mechanism for providing help to generate new specificities of alloantibody in transplant patients receiving immunosuppression.

is a professor of chemical engineering at Bucknell University. She earned her B.S. in chemical engineering from Cornell University, and her M.S. and Ph.D., also in chemical engineering, from the University of Virginia. Her primary research focus is on engineering pedagogy at the undergraduate level. She is particularly interested in the teaching and learning of concepts related to thermodynamics. She is also interested in active, collaborative, and problem-based learning, and in the ways hands-on activities such as making, technology, and games can be used to improve student engagement.

She holds a joint appointment as Research Associate Professor in the Department of Mechanical Engineering and the School of Education and Social Policy. She also serves as co-director of the Northwestern Center for Engineering Education Research (NCEER). Dr. McKenna's research focuses on understanding the cognitive and social processes of design teaching and learning, the role of adaptive expertise in design and innovation, and teaching approaches of engineering faculty. Dr. McKenna received her B.S. and M.S. degrees in Mechanical Engineering from Drexel University and Ph.D. in Engineering, Science and Mathematics Education from the University of California at Berkeley.

has a B.S. in biomedical engineering from Florida International University (Fall 2010) and is planning to pursue a MS in biomedical engineering in the upcoming year. Mr. Molina assisted with the data analysis in this study as an REU student, with an interest in assesment and educational researchy.

Graduate Program. Formerly, he was the Associate Director of the VaNTH Engineering Research Center in Bioengineering Educational Technologies, and chair of the Biomedical Engineering Department at Northwestern. He is a fellow of the American Institute of Medical and Biological Engineering, the Biomedical Engineering Society, and the Association for Research in Vision and Ophthalmology. His research interests are in the role of retinal oxygen transport and metabolism in both normal physiological conditions and disease, and in bioengineering and physiology education.

is a professor of chemical engineering at Bucknell University. She earned her B.S. in chemical engineering from Cornell University, and her M.S. and Ph.D., also in chemical engineering, from the University of Virginia. Her primary research focus is on engineering pedagogy at the undergraduate level. She is particularly interested in the teaching and learning of concepts related to thermodynamics. She is also interested in active, collaborative, and problem-based learning, and in the ways hands-on activities such as making, technology, and games can be used to improve student engagement.