B cells help alloreactive T cells differentiate into memory T cells

Abstract

B cells are recognized as effector cells in allograft rejection that are dependent upon T cell help to produce alloantibodies causing graft injury. It is not known if B cells can also help T cells differentiate into memory cells in the alloimmune response. We found that in B-cell-deficient hosts, differentiation of alloreactive T cells into effectors was intact whereas their development into memory T cells was impaired. To test if B cell help for T cells was required for their continued differentiation into memory T cells, activated T cells were sorted from alloimmunized mice and transferred either with or without B cells into naïve adoptive hosts. Activated T cells cotransferred with B cells gave rise to more memory T cells than those transferred without B cells and upon recall, mediated accelerated rejection of skin allografts. Cotransfer of B cells led to increased memory T cells by enhancing activated CD4 T-cell proliferation and activated CD8 T-cell survival. These results indicate that B cells help alloreactive T-cell differentiation, proliferation and survival to generate optimal numbers of functional memory T cells.

Figure 1. Development of alloreactive memory T cells is impaired in B-cell deficient mice

A. Rejection of BALB/c (H-2^d) skin allografts transplanted to µMT (H-2^b) and wt (H-2^b) mice (MST = 16 and 15 days, respectively; p > 0.05; n = 10 mice/grp). Quantitiation of alloreactive effector (B) and memory (D) T cells in µMT and wt mice. Spleen (SP), lymph node (LN) and bone marrow (BM) cells were harvested from BALB/c skin grafted µMT and wt recipients at 14-days (effector phase) and at 8-weeks after rejection (memory phase) for quantitation of alloreactive T cells. Harvested cells were re-stimulated ex-vivo for 6-hrs with BALB/c splenocytes and IFNγ producing CD4 and CD8 T cells were assessed by flow cytometry and enumerated (Mean ± SD; *, p < 0.05; n = 4 mice/grp). C. CD8 memory precursors within alloreactive effector T cells in µMT and wt mice. Representative FACS plots of IL-7Rα expression on BALB/c-reactive IFNγ⁺ population within CD8⁺ CD44^hi splenic T cells harvested at 14-days after BALB/c skin transplantation are shown. E–F. Cytotoxic function of µMT and wt memory T cells was assessed by in-vivo (E) and in-vitro (F) allogeneic cell lysis at 8-weeks after BALB/c skin graft rejection (memory phase). µMT and wt mice were depleted of NK cells and equal numbers of CFSE labeled H-2^b (2 × 10⁷, 2µM B6 wt, syngeneic) and H-2^d (2 × 10⁷, 0.2µM, BALB/c, allogeneic) splenocytes were injected (i.v.). 24-hrs later, in-vivo killing of BALB/c cells in comparison to B6 cells was measured by flow cytometry (Mean ± SD; **, p < 0.005; n = 3 – 4 mice/grp). Purified T cells from SP and LN cells of µMT and wt memory mice were incubated with calcein labeled BALB/c splenocytes (0.3mM, 100:1) and 4-hrs later, in-vitro killing of BALB/c cells was measured by flow cytometry (Mean ± SD; *, p < 0.05; n = 3 –4 mice/grp). G. Allograft rejection in memory µMT and wt mice. At 8-weeks after BALB/c allograft rejection (memory phase), µMT and wt recipients were re-challenged with BALB/c skin grafts and treated with DST (2 × 10⁷) and anti-CD40L (MR1, 1mg on days 0, 7 and 14 after transplantation). Allograft rejection was assessed in µMT and wt memory mice, and compared to naïve mice (MST = 30, 20 and 30 days, respectively; p < 0.05; n = 4 – 9 mice/grp).

Figure 2. B cells promote differentiation of activated T cells to memory T cells in adoptive hosts

CD45.1 mice were immunized with BALB/c splenocytes (3 × 10⁷) and 8-days later, SP and LN cells were harvested and sorted for CD4⁺ and CD8⁺ CD44^hi T cells and B220⁺ B cells. Sorted CD4⁺ or CD8⁺ CD44^hi activated T cells (1 × 10⁶) were transferred (i.v.) with or without B220⁺ B cells (1.5 × 10⁷) into µMT and wt mice. SP, LN and BM cells from µMT and wt hosts were harvested at 1, 2, 3 and 8–12 weeks (C and D, respectively) after adoptive transfer and were enumerated after gating on CD4⁺ or CD8⁺ CD45.1⁺ T cells. A. CD45.1⁺ T and B cells in µMT adoptive hosts. Representative FACS plots from spleen cells harvested at 10-weeks after transfer from µMT recipients of activated T cells transferred with or without B cells are shown. y-axis legend is depicted on top of the FACS plots. B. Memory phenotype of CD45.1⁺ T cells harvested from adoptive hosts at 10-weeks after transfer. Representative FACS plots are shown from spleen cells of µMT adoptive recipient of CD8⁺ CD44^hi activated T cells co-transferred with B220⁺ B cells. y-axis legend is depicted on top of the FACS plots. C. Activated T cells transferred without B cells decline over time in adoptive hosts (Mean ± SD, n = 2 – 3 mice/grp). D–E. Activated T cells co-transferred with B cells develop into more memory T cells in adoptive hosts. B220⁺ B cells were sorted from either CD45.1 mice immunized with BALB/c splenocytes at 8-days (D) or from unimmunized naïve CD45.1 mice (E) (Mean ± SD; *, p < 0.05, **, p < 0.005; n = 4 – 6 mice/grp).

Figure 3. Proliferation and survival of activated T cells in adoptive hosts

Activated CD4⁺ or CD8⁺ CD44^hi T cells and B220⁺ B cells were sorted from SP and LN cells of immunized CD45.1 mice (BALB/c splenocytes, 3 × 10⁷) at 8-days. Sorted CD45.1 CD4⁺ and CD8⁺ CD44^hi T cells (1 × 10⁶) were CFSE labeled and transferred with or without B220⁺ B cells (1.5 × 10⁷) into µMT and wt adoptive hosts. At 2-weeks after transfer, CFSE dilution was analyzed on harvested SP and LN cells after gating on CD4⁺ or CD8⁺ CD45.1⁺ T cells. Representative histogram overlays of CFSE dilution in CD4⁺ or CD8⁺ CD45.1⁺ T cells from harvested spleens of µMT and wt adoptive hosts are shown (A). B. B cells help proliferation of activated CD4 T cells in adoptive hosts. Proliferation index is calculated using Flowjo software after gating on CD4⁺ or CD8⁺ CD45.1⁺ T cells (Mean ± SD; *, p < 0.05; n = 3 – 4 mice/grp). C. Apoptosis of activated T cells in adoptive hosts. Harvested SP and LN cells from µMT and wt adoptive hosts were examined for Annexin V⁺ staining after gating on CD4⁺ or CD8⁺ CD45.1⁺ T cells (Mean ± SD; n = 3 – 4 mice/grp). D. Bcl-2 expression in CD4⁺ and CD8⁺ CD45.1⁺ T cells in adoptive hosts. Representative histogram overlays of Bcl-2 expression are shown after intracellular staining and gating on harvested CD4⁺ or CD8⁺ CD45.1⁺ T cells from adoptive hosts. E. Bcl-2 expression is upregulated in developing CD8 memory T cells in adoptive hosts harboring co-transferred B cells. Median fluorescence intensity of Bcl-2 expression on CD8⁺ CD45.1⁺ T cells harvested from adoptive hosts is shown in comparison to isotype control (Mean ± SD; *, p < 0.05, n = 3 – 4 mice/grp).

Figure 4. Allograft rejection in adoptive hosts harboring memory T cells

Memory T cells that developed from activated CD4⁺ or CD8⁺ CD45.1⁺ T cells co-transferred with or without B220⁺ B cells into adoptive hosts were re-challenged with BALB/c skin grafts at 12-weeks after adoptive transfer (memory phase). Skin allograft recipients were treated with DST (2 × 10⁷) and anti-CD40L (MR1, 1mg on days 0, 7 and 14 after transplantation). Allograft rejection in µMT and wt adoptive hosts harboring memory T cells was compared to naïve mice. MST in wt naïve and adoptive hosts of activated CD4 vs. CD4 + B, CD8 vs. CD8 + B cells were 33, 35 vs. 25 and 32 vs. 24 days, respectively (**, p < 0.005; n = 4 – 9 mice/grp). MST in µMT naïve and adoptive hosts of activated CD4 vs. CD4 + B, CD8 vs. CD8 + B cells were 30, 26 vs. 20 and 28 vs. 19 days, respectively (**, p < 0.005; n = 4 – 9 mice/grp).

Figure 5. Model of B cell help for T cell differentiation to memory

Early in the immune response professional APCs such as dendritic cells (DCs) present antigen to T cells and provide early co-stimulatory signals. As antigen specific B and T cells proliferate, the non-dividing DCs become limiting to support further differentiation of T cells. B cells then take over as ‘helper’ cells to promote continued differentiation and survival of T cells. B cells present entrapped antigen, provide cytokines and/or co-stimulation to T cells to support their differentiation into long-lived memory T cells.