T follicular helper cells expansion in transplant recipients correlates with graft infiltration and adverse outcomes

Abstract

Introduction: The process of immunization following vaccination in humans bears similarities to that of immunization with allografts. Whereas vaccination aims to elicit a rapid response, in the transplant recipient, immunosuppressants slow the immunization to alloantigens. The induction of CD4+CXCR5+ T follicular helper (Tfh) cells has been shown to correlate with the success of vaccine immunization.

Method: We studied a cohort of 65 transplant recipients who underwent histological evaluation concurrent with PBMC isolation and follow-up sampling to investigate the phenotypic profiles in the blood and allotissue and analyze their association with clinical events.

Results: The proportion of circulating Tfh cells was heterogeneous over time. Patients in whom this compartment increased had lower CCR7-PD1+CD4+CXCR5+ T cells during follow-up. These patients exhibited more alloreactive CD4+ T cells using HLA-DR-specific tetramers and a greater proportion of detectable circulating plasmablasts than the controls. Examination of baseline biopsies revealed that expansion of the circulating Tfh compartment did not follow prior intragraft leukocyte infiltration. However, multicolor immunofluorescence microscopy of the grafts showed a greater proportion of CXCR5+ T cells than in the controls. CD4+CXCR5+ cells were predominantly PD1+ and were in close contact with B cells in situ. Despite clinical stability at baseline, circulating Tfh expansion was associated with a higher risk of a composite of anti-HLA donor-specific antibodies, rejection, lower graft function, or graft loss.

Conclusion: In otherwise stable patients post-transplant, circulating Tfh expansion can identify ongoing alloreactivity, detectable before allograft injury. Tfh expansion is relevant clinically because it predicts poor graft prognosis. These findings have implications for immune surveillance.

Keywords: anti-HLA antibodies; follicular helper T cells; graft outcomes; kidney transplantation; rejection.

Figure 1

The heterogeneity in the circulating CD4+CXCR5+ pool over time correlates with variations in PD1 and ICOS expression. We classified the patients of the cohort according to the evolution in the expression of CXCR5+ in CD4+ T cells at baseline and at 3 months. (A) Representative examples of CXCR5 expression in CD4+ T cells across tertiles. (B) CD4+CXCR5+ counts in the blood. Each individual is illustrated as a single line. (C) Ratios of month 3 versus month 0 according to tertiles. (D) Expression of PD1 in CD4+CXCR5+ T cells for each individual by tertile (left) and summary ratios across tertiles (right). (E) Proportion of CCR7-PD1+ in CD4+CXCR5+ T cells across tertiles. (F) Proportion of ICOS+PD1+ in CD4+CXCR5+ T cells across tertiles. Left, comparison of M0 versus M3 for each individual illustrated as a single line, by tertile. Right, comparison between tertiles at M0. (G) Dotplots depicting the relationship between CD4+CXCR5+PD1+ M3/M0 ratio versus CD4+CXCR5+ICOS+ M3/M0 ratio on an individual basis; each dot illustrates an individual. M0, month 0; M3, month; n=65 for all subfigures.

Figure 2

Patients with an expansion of circulating CD4+CXCR5+ T cells present with more circulating HLA-specific alloreactive T cells, plasmablasts and intragraft Tfh cells. (A) Proportion of CD4+tetramer+ cells according to the absence or presence of circulating CD4+CXCR5+ T cells increase at 3 months (n=17). (B) Corresponding blood cells counts (n=17). (C) Baseline proportion of patients with detectable circulating CD19+CD27+CD38hi plasmablasts according to tertiles of circulating CD4+CXCR5+ T cells expansion (n=61). (D) Proportion of patients at 3 months for the same cell population (n=63). (E) Proportion of CXCR5+ positive cells in the CD3+ compartment in biopsies, according to the absence or presence of circulating CD4+CXCR5+ T cells increase at 3 months (n=32). (F) Multicolor immunofluorescence of CD4, CXCR5, and PD1 in a renal allograft. (G) Proportion of PD1+ cells in the CD4+CXCR5+ compartment in the allografts versus in the blood n=12). (H) Multicolor immunofluorescence of CD4, CXCR5, and CD19 in a renal allograft. M0, month 0; M3, month 3; Undet, undetectable; Det, detectable.

Figure 3

Expansion of circulating CD4+CXCR5+ T cells is not related to prior signs of activation in the allograft, but is associated with poorer clinical outcomes. The compartments assessed for rejection included (A) the tubulointerstial compartement: leucocyte infiltration in the tubules in unscared area (tubular infiltration, t score), the interstitium unscared area (interstitial inflammation, i score), the interstitium in both scarred and unscarred area (total interstitial inflammation, ti score) and (B) the microvascular compartment: the glomerular capillaries (glomerulitis, g score), and the peritubular capillaries (peritubular capillaritis, ptc score). In addition, the deposition of the C4d component of the complement was assessed (C4d score) as an indirect evidence of antibody ligation to the endothelium in the peritubular capillaries, witnessing upstream C1q activation by antibodies. (C) Boxplots and individual values of eGFR post-enrolment. (D) Longitudinal eGFR by tertile of circulating CD4+CXCR5+ T cell ratios at 3 months versus baseline; each line illustrates an individual. (E) Kaplan-Meier curve of the composite endpoint by tertile. (F) Cox models for the composite endpoint, unadjusted and adjusted for recipient age and sex, first transplant, time post transplant, eGFR at first blood sample, and donor type. eGFR, estimated glomerular filtration rate; HR, hazard ratio; n=65 for all subfigures.