Persistence of long-lived plasma cells and humoral immunity in individuals responding to CD19-directed CAR T-cell therapy

Abstract

The mechanisms underlying the maintenance of long-lasting humoral immunity are not well understood. Studies in mice indicate that plasma cells (PCs) can survive up to a lifetime, even in the absence of regeneration by B cells, implying the presence of long-lived PCs as a mechanism for long-lasting immunity. Evidence from humans treated with anti-CD20, which depletes circulating B cells, also suggests B-cell-independent long-term survival of some PCs. On the other hand, antibody responses may be sustained solely by short-lived PCs with repopulation from clonally related memory B cells. To explore PC longevity and humoral immunity in humans, we investigated the fate of PCs and their antibodies in adult and pediatric patients who received chimeric antigen receptor-based adoptive T-cell immunotherapy targeting CD19 to treat B-cell lineage malignancies (CTL019). Treatment with CTL019 is frequently associated with B-cell aplasia that can persist for years. Serum antibody titers to vaccine-related antigens were measured, and quantitative assessment of B cells and PCs in blood and bone marrow was performed at various time points before and after CTL019 therapy. While total serum immunoglobulin concentrations decline following CTL019-induced B-cell aplasia, several vaccine/pathogen-specific serum immunoglobulin G and A (IgG and IgA) titers remain relatively stable for at least 6 and 12 months posttreatment, respectively. Analysis of bone marrow biopsies after CTL019 revealed 8 patients with persistence of antibody-secreting PCs at least 25 months post-CTL019 infusion despite absence of CD19(+)CD20(+) B cells. These results provide strong evidence for the existence of memory B-cell-independent, long-lived PCs in humans that contribute to long-lasting humoral immunity.

Figure 1

Scheme of subject selection and testing. Samples from patients enrolled in CTL019 clinical trials UPCC04409, UPCC13413, and CHP959 were tested for the presence of B cells, PCs, and serum antibodies to evaluate the state of humoral immunity posttreatment. H&E, hematoxylin and eosin.

Figure 2

CD19⁻ bone marrow PCs resist elimination by CTL019. (A) Bone marrow aspirate from healthy donors were stained and analyzed by flow cytometry. The bottom right panel shows CD19 expression on CD138⁺CD38⁺ PCs (red) and CD20⁺CD19⁺ cells (blue); the green histogram represents CD138⁺CD38⁺ cells with fluorescence-minus-one (CD19) staining. Representative data from 7 donors are shown. The “dump” channel contains stains for viability, CD3, CD14, and CD16. (B) Bone marrow aspirate cells from CHP-11 on day 28 post-CTL019 infusion were analyzed as in panel A. Gating for B cells (top left), PCs (top right) is shown. The bottom left panel shows CD19 expression on CD38⁺CD138⁺ cells (red histogram) and on all singlet events in the forward (FSC) and side scatter (SSC) gate (blue histogram). In both panels A and B, expression of light chains was assessed by intracellular staining. (C) Two hundred thousand cells from the aspirate material shown in panel B were plated in ELISPOT wells, which were stained for total IgG, IgM, and IgA after overnight incubation.

Figure 3

Immunohistochemical analysis demonstrates long-term presence of bone marrow PCs and B-cell aplasia after CTL019 treatment. (A) Bone marrow core biopsies from subject UPN-1 at baseline and serial time points post-CTL019 infusion were stained, separately, for CD138, CD20, and CD19 by IHC. Representative regions at ×20 magnification are shown. (B-E) The frequency of CD138⁺ (B), CD20⁺ (C), and CD19⁺ cells (D) among total nucleated cells in bone marrow biopsies from adult (red curves) and pediatric (blue curves) patients was determined by digital image analysis for the indicated time points pre- and post-CTL019.

Figure 4

CTL019 results in B-cell depletion in primary and secondary lymphoid tissues. Sections of bone marrow, spleen, lymph nodes, terminal ileum, and colon, obtained at autopsy from a CTL019-treated subject (day 234), were analyzed by H&E and IHC (CD20, κ, λ). Representative sections are shown at ×20 (all spleen and CD20 images) and ×40 magnification (all other images). H&E insets are shown at ×100 magnification.

Figure 5

Antigen-specific antibody titers at baseline and after CTL019 infusion. (A) S pneumoniae–, (B) H influenzae type B–, (C) tetanus toxoid–, (D) measles-, (E) mumps-, and (F) rubella-specific IgG, and (G) HSV1/2-specific IgA levels were measured in CTL019-treated patients who experienced persistent B-cell aplasia. For anti-H influenzae, -tetanus toxoid, -rubella, and anti-measles, thresholds of protective IgG levels are indicated by the dashed lines. For anti-S pneumoniae, anti-mumps, and anti-HSV1/2 IgA, the dashed lines indicate the lower limit of detection. Error bars indicate standard deviations.

Figure 6

CD19⁻ PCs produce vaccine/pathogen-specific antibodies. (A) Bone marrow aspirate cells from healthy donors were sorted into 4 populations (CD19⁺ PCs, CD19⁻ PCs, B cells, and Dump⁺) and were analyzed by ELISPOT. (B) Total IgG, tetanus toxoid–, measles-, or mumps-specific IgG-producing cells were enumerated by ELISPOT. Each symbol represents an individual donor. Means and standard deviations are indicated. For total IgG, all pairwise comparisons demonstrated a P value of .0625 except CD19⁺ PC vs CD19⁻ PC and B cells vs Dump. For tetanus toxoid, all pairwise comparisons demonstrated a P value of .0625 except B cells vs Dump. For measles, only CD19⁺ PC vs B cells and CD19⁺ vs Dump comparisons demonstrated a P value of .0625. P values for all other pairwise comparisons were .1088 or greater (Wilcoxon signed-rank test). ASC, antibody-secreting cells.