Fig 2: Comparable VH and JH Intron Mutation Frequencies in B Cells of Acutely and Chronically Infected Mice(A–C) We performed adoptive transfer experiment as in Figure 4, and on day 36, we sorted isotype-switched adoptively transferred KL25HL-AIDrep GC B cells (CD45.1+ CD45.2–IgM–IgD–GL7+B220+) by FACS for IgH locus sequencing. (A) Mutation frequencies in the JH intron as base pair (bp) changes are shown for all sequences obtained (left) as well as for mutated introns only (right). (B) Mutations in the KL25 VH gene as total amino acid changes per V sequence, for all sequences (left) or sequences with amino acid changes (right), taking only functional sequences into account. (C) All amino acid mutations collected from four rARM*- and four rCl13*-infected mice (708 sequences total, 85–91 sequences per mouse) are represented along the KL25 VH protein sequence (top: rCl13* infection; bottom: rARM* infection). Mutations are shown in bold red letters when present in at least four individual mice (out of eight). CDR positions are shaded in gray. Bars show means ± SD, and symbols represent individual mice. n = 4, N = 1 (A–C). Unpaired two-tailed Student’s t test (A and B). ns, p = 0.05.(D) Binding of KL25 wild-type and KL25-W104L to WE-GP and WE-GP*.(E) Neutralization of rCl13 and rCl13* by KL25 wild-type and KL25-W104L. Symbols in (D) and (E) show the mean of 2 technical replicates. N = 3 (D and E). See also Figure S5.

Fig 3: rhIL-7-hyFc enhances mCART19 expansion and persistence in immunocompetent mice.a C57BL/6J mice were injected with 5 × 104 syngeneic tumor cells (9523, a myeloid cell line to which murine CD19 expression was added) followed by cyclophosphamide (Cytoxan) conditioning one day prior to receiving 6 × 106 UTD or murine CART19 (mCART19), then given serial injections of rhIL-7-hyFc on days +1, +15, and +29 (n = 4/group). b Overall survival of mice in each group. p Values were calculated using two-sided Wilcoxon test (mCART19 + rhIL-7-hyFc vs. mCART19, p = 0.32, mCART19+rhIL-7-hyFc vs. UTD + rhIL-7-hyFc, p = 0.044). c Peripheral blood total WBC counts over time. d Absolute numbers of mCART19 cells over time. e Total WBC counts were fractionated by flow cytometry into percent CD45.1+ (adoptively transferred T cells) and CD45.2+ (endogenous cells); CD45.2 cells are subdivided into CD19+ B cells, CD3+ T cells, and Gr-1+ myeloid cells at the indicated timepoints. f Absolute numbers of CD45.2+ B cells, T cells, and myeloid cells over time, calculated by multiplying the total WBC count by the fraction of each cell determined by flow cytometry. All data represent median ± range. Dotted lines represent rhIL-7-hyFc injection time points. Source data are provided as a Source data file. ns: not significant, *p = 0.05.

Fig 4: Chronic Infection Is a Potent Long-Term Driver of B Cell Responses(A) We infected syngeneic recipient mice with rARM* or rCl13* on day 0, and 20 days later, we adoptively transferred CTV-labeled KL25HL-AIDrep B cells. TAM was administered on the day of transfer (day 20) and on day 23.(B and C) Five and 15 days after adoptive transfer (day 25 and day 35), we analyzed KL25HL-AIDrep B cells by flow cytometry for AID reporting (EYFP+) and GC differentiation (GL7 expression). Representative FACS plots in (B) are gated on adoptively transferred proliferated B cells (CD45.1+ CD45.2- CTVlo B220+ CD138- cells, gating strategy in Figure S4). The average proportional representation of EYFP+GL7– (red), EYFP+GL7+ (orange), EYFP–GL7+ (magenta), and EYFP–GL7– (blue) subsets is displayed in pie charts. Absolute numbers of these same cell populations are shown in (C). Bars represent means ± SEM, n = 3–5.(D) The concentration of KL25 IgG in the serum of KL25HL-AIDrep B cell recipients was determined by GP-1 ELISA. Background GP-1-specific IgG levels in control mice without KL25HL-AIDrep B cell transfer were at least 4-fold lower than in the respective groups of recipients. Symbols show individual mice.(E) Histological sections from spleens of rCl13* and rARM* infected mice on day 25. Scale bar: 50 µm. n = 3, N = 2.Two-way ANOVA with Bonferroni’s post-test for multiple comparisons (C), unpaired two-tailed Student’s t test (D). *p < 0.05, **p < 0.01. See also Figure S4.

Fig 5: In vivo rhIL-7-hyFc therapy enhances both the quantity and quality of CAR T cells.a–g Balb/c mice were given cyclophosphamide 250 mg/kg on day -1, then 2 × 106 CD45.1+ UTD or mCART19 cells on day 0, followed by rhIL-7-hyFc on day +1. Spleens were harvested on days +7 and +14 after T cell injection (n = 3/group). Bar graphs represent median values, with each data point representing one mouse. a Spleen sizes (left, day +14) and cell numbers (right) were significantly increased in mice given rhIL-7-hyFc. b Fractionation of spleen cell subsets shows that the majority of cells are derived from the CD45.2+ recipient. c Absolute numbers of mCART19 cells were higher in the spleens of mice after rhIL-7-hyFc treatment. d Apoptosis was reduced in mCART19 at day +14 with rhIL-7-hyFc. e Both UTD and mCART19 show marked proliferation in response to rhIL-7-hyFc on day +7, which is better sustained at day +14 for mCART19. f, g On day +7 mCART19 was purified from spleens and subject to in vitro functional assays. f mCART19 cytotoxicity against A20 tumor cells was measured at various E:T ratios by BLI after 48 h of incubation. Each line represents mCART19 cells extracted from one mouse. g Intracellular IFN? and TNFa production of mCART19 was measured after 4 h of incubation with A20 tumor cells. h-j NSG mice received 5×105 RamosCBR-GFP followed by UCART19 treatment and rhIL-7-hyFc on days +1 and +15. Spleens were harvested from mice on day +28. h Spleens harvested from rhIL-7-hyFc-treated mice were notably larger than controls. i, j Target cell viability was determined by FACS-based cytotoxicity assay of UCART19 from day 0 (input) as compared to UCART19 derived from spleens of rhIL-7-hyFc treated mice. The original Ramos tumor cells (i) or an independent CD19+ B cell line, Nalm6 cells (j) were incubated with CD34-purified UCART19 at E:T ratios ranging from 4:1 to 1:4 for 20 h. Bar graphs represent median values, with each data point representing a technical replicate. All p values were determined by two-sided unpaired Student’s t test. p Values and source data are provided in the Source data file. ns: not significant, *p = 0.05, **p = 0.01, ***p = 0.001, ****p = 0.0001.