Evidence for CD19+ (B) CD8+ (T)-Cell Interactions in Blood and Tissues from Patients with Graft-Versus-Host Disease
Authors: Deola Sara , Cugno Chiara , Comoli Patrizia , Guido Ilaria , Rubert Laura , Zecca Marco , Zanolini Diego , Follenzi Antonia , Langes Martin , Negri Giovanni , Tauber Martina , Pusceddu Irene , Cavattoni Irene , Svaldi Mirija , Wang Ena , Rutella Sergio , Marincola Francesco M 
Published: October 2019

Abstract
Graft-versus -host disease (GVHD), a major complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT), is mediated by cytotoxic T cells (CTL), recruited to target organs by immune-mediated injury. There is growing evidence that B cells play a pathogenic role in GVHD, although the underlying mechanisms are unclear.
We previously described the interaction between autologous CD19+ B cells and activated CD8+ CTLs in normal donors (Deola et al, 2008). In vitro antigen-stimulated CTLs formed stable couplets with B cells, independent of antigen presentation. Coupling enhanced CTL survival and proliferation, and stimulated B-cell release of chemokines, such as MIG, IP-10 and I-TAC, which are pivotal for T-cell migration to GVHD target organs.
Herein, we retrospectively analyzed a cohort of patients undergoing immune monitoring after allo-HSCT. To determine the role of CD19(B)-CD8(T) coupling in GVHD, we purposely included patients with both acute and chronic GVHD who had at least 5% circulating B cells. The cohort included 292 consecutive blood samples: 176 from GVHD patients (acute, chronic, overlap syndrome), 116 from non-GVHD patients, and 21 from healthy controls. We restricted sample selection by including only patients with GVHD onset within 150 days from sampling (26 patients, 40 samples overall), and we excluded from the non-GVHD sample cohort any patient with previous or suspect GVHD (achieving 36 samples overall, belonging to 15 different patients).
B-T couplets were significantly higher in patients with GVHD, compared with both allo-HSCT patients without GVHD and with healthy controls (p=0.048 and 0.0008, respectively). Particularly remarkable was the difference between patients developing GVHD within 30 days from sampling and the controls. B-T couplets were measured in 7 patients where sampling was available both before and after GVHD onset, and the difference was again statistically significant (p=0.016).
Nine patients in the entire cohort received donor lymphocyte infusions (DLI) for a molecular relapse of their disease. Interestingly, B-T couplets were significantly higher after DLIs, regardless of subsequent GVHD development.
A validation cohort of children with acute or chronic GVHD after allo-HSCT was then analyzed. This consisted of 13 patients with no signs of GVHD and 11 patients with GVHD. Patients with GVHD had a significantly higher number of B-T couplets compared with non-GVHD patients (p=0.029). By focusing the analysis on patients with GVHD onset within 150 days prior to immune monitoring (7 patients), the p-value for the comparison was 0.014. Again, the score of couplets in patients developing GVHD within 30 days from sampling was higher.
We finally looked at tissue distribution of B and T cells in 7 skin and gut biopsies from patients with GVHD, obtained 3-13 months after allo-HSCT. Three skin biopsies of allo-HSCT patients without features of GVHD were also included.
Immunohistochemistry for CD20 showed that 5 out of 7 biopsies from patients with GVHD hosted B cells (up to 10), focally coupled with CD8+ T cells. The control biopsies from patients without signs of GVHD 4-15 months after allo-HSCT (n=3) stained negatively for B cells. GVHD and control biopsies were also screened with laser scanner microscope, again finding evidence of B-cell/T-cell coupling.
Collectively, these data point to the interplay between B cells and CTL both in blood and in tissues from allo-HSCT patients with GVHD, as well as after DLI. Conceivably, B-T cell coupling amplifies the inflammatory signal, both systemically and locally. B cells may attract additional immune cells, thus supporting CTL survival and expansion.

Disclosures
No relevant conflicts of interest to declare.