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. Author manuscript; available in PMC: 2017 Nov 17.
Published in final edited form as: Cell Rep. 2017 Nov 14;21(7):1839–1852. doi: 10.1016/j.celrep.2017.10.077

Th1-like Plasmodium-specific memory CD4+ T cells unexpectedly support humoral immunity

Ryan A Zander 1,8, Rahul Vijay 7, Angela D Pack 7, Jenna J Guthmiller 1,9, Amy C Graham 1, Scott E Lindner 3,4,5, Ashley M Vaughan 5, Stefan H I Kappe 5,6, Noah S Butler 1,2,7,*
PMCID: PMC5693336  NIHMSID: NIHMS915564  PMID: 29141217

Summary

Effector T-cells exhibiting features of either T helper 1 (Th1) or T follicular helper (Tfh) populations are essential to control experimental Plasmodium infection and are believed to be critical for resistance to clinical malaria. To determine whether Plasmodium-specific Th1- and Tfh-like effector cells generate memory populations that contribute to protection, we developed transgenic parasites that enable high-resolution study of anti-malarial memory CD4 T-cells in experimental models. We found that populations of both Th1- and Tfh-like Plasmodium-specific memory CD4 T-cells persist. Unexpectedly, Th1-like memory cells exhibit phenotypic and functional features of Tfh cells during recall and provide potent B-cell help and protection following transfer, characteristics that are enhanced following ligation of the T-cell co-stimulatory receptor OX40. Our findings delineate critical functional attributes of Plasmodium-specific memory CD4 T-cells and identify a host-specific factor that can be targeted to improve resolution of acute malaria and provide durable, long-term protection against Plasmodium parasite re-exposure.

eTOC Blurb

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Th1 CD4 T cells are widely described as terminally differentiated with a relatively reduced capacity to form memory or support humoral immunity. Using experimental malaria models, Zander et al. show that potent proliferative and B cell helper activity unexpectedly resides within the Plasmodium-specific Th1-like memory CD4 T cell compartment.

Introduction

Plasmodium infections cause over 300 million cases of malaria and claim more than 400,000 lives annually (Organization, 2015). Although clinical immunity against the development of severe malaria can be established following repeated exposure, sterilizing immunity that protects individuals against re-infection rarely develops (Portugal et al., 2013, Crompton et al., 2014). Immune-mediated protection against repeated infection generally requires the activity of memory lymphocytes that develop following either primary infection or after vaccination (Jaigirdar and MacLeod, 2015, McKinstry et al., 2010). Elevated numbers of memory CD4 T cells are a hallmark of adaptive immune memory (Pepper and Jenkins, 2011). In addition to larger precursor frequencies, the capacity of memory CD4 T cells to rapidly control infection is associated with their enhanced trafficking patterns (Mueller et al., 2013), cytokine expression (Chandok et al., 2007), and ability to support humoral immunity (He et al., 2013). While adaptive immune memory is important for resistance to re-infection, the factors that govern memory CD4 T cell development, differentiation, and function remain less well understood. Moreover, one hypothesis to explain persistent and recurrent susceptibility to Plasmodium infection is that parasite-specific memory CD4 T cell populations are either numerically or functionally deficient. Addressing this question has been difficult, however, given the paucity of reagents necessary to study anti-Plasmodium CD4 T cell memory. Thus, the quantitative and qualitative features and mechanisms regulating the development of Plasmodium-specific memory CD4 T cells remain major knowledge gaps.

Resistance to clinical malarial disease is believed to require CD4 T cells exhibiting features of either T helper 1 (Th1) or T follicular helper (Tfh) cells (Jagannathan et al., 2014, Jagannathan et al., 2015, Obeng-Adjei et al., 2015). Plasmodium infection-induced Th1 cells secrete IFN-γ and stimulate macrophage phagocytic function respiratory burst, which aids in elimination of parasites and parasite-infected red blood cells (von der Weid and Langhorne, 1993). Tfh cells induce long-lived memory B cell and antibody-secreting plasma cell responses (Crotty, 2014). Experimental models have established the essential role of Th1 and Tfh cells in mediating host resistance to malaria (Carpio et al., 2015, Freitas do Rosario and Langhorne, 2012, Gwyer Findlay et al., 2014, Opata et al., 2015, Perez-Mazliah et al., 2015, Stephens et al., 2005, Stephens and Langhorne, 2010, Villegas-Mendez et al., 2013). In addition to secretion of IFN-γ, Th1 cells are characterized by expression of the transcriptional regulator T-bet (Szabo et al., 2000), lymphocyte antigen 6C (Ly6C) (Marshall et al., 2011, Yamanouchi et al., 1998), and the chemokine receptor CXCR3 (Sallusto and Lanzavecchia, 2000). Tfh cells are identified by expression of CXCR5 (Kim et al., 2001) and PD-1 (Haynes et al., 2007), secretion of IL-21 (Chtanova et al., 2004), and are regulated by the transcriptional repressor Bcl-6 (Crotty, 2011, Choi et al., 2013). An emerging literature supports that Plasmodium infection-induced CD4 T cell populations can exhibit mixed profiles that reflect both Tfh- and Th1-like phenotype and function. For example, Plasmodium infection-induced effector CD4 T cells can co-express effector cytokines IFN-γ and IL-21 (Carpio et al., 2015, Freitas do Rosario et al., 2012, Perez-Mazliah et al., 2015, Ryg-Cornejo et al., 2016) and effector CXCR5+CXCR3+ Tfh cells are preferentially expanded in P. falciparum-infected children during acute malaria (Obeng-Adjei et al., 2015).

The study of memory CD4 T cell responses against Plasmodium has been hampered by the lack of defined parasite-derived T cell epitopes that are necessary for their identification and interrogation of function. Thus, whether distinct Plasmodium-specific Th1- and Tfh-like memory CD4 T cell subsets either persist, exhibit plasticity during recall responses, or differentially support protective immunity are not well known. To overcome these obstacles and address these questions we generated transgenic P. yoelii parasites engineered to encode the hepatocyte-erythrocyte protein of 17kDa (Hep17) tagged with a dominant CD4 T cell epitope from lymphocytic choriomeningitis virus (LCMV). Hep17 is found in the parasitophorous vacuole, host cytoplasm, and on the surface of merozoites (Charoenvit et al., 1995), and immunization with recombinant Hep17 stimulates protective immunity (Charoenvit et al., 1999). With these reagents, we identified that both Th1- and Tfh-like Plasmodium-specific memory CD4 T cells form, persist, and can be distinguished by differential expression of Ly6C and CXCR5. Plasmodium-specific memory CD4 T cells within the CXCR3+Ly6C+ Th1-like subset exhibit unanticipated potent secondary expansion, protective capacity, and superior B cell helper function, features that were each enhanced by therapeutic ligation of the co-stimulatory molecule OX40 during acute malarial disease.

Results

Therapeutic ligation of OX40 during acute P. yoelii infection increases the number of parasite-specific memory CD4 T cells

To determine the magnitude and kinetics of the parasite-specific CD4 T cell response during experimental malaria we infected groups of wild type C57BL/6 (WT B6) mice with recombinant P. yoelii parasites (Py-GP) engineered to express the immunodominant CD4 T cell epitope from LCMV (amino acids 61–80 of the glycoprotein, GP61–80; Fig. S1A,B). Two independent isolates of Py-GP each exhibited growth kinetics that closely match the parental strain (Fig. S1C). Given the dramatic increase in the expansion of effector CD4 T cells upon therapeutic ligation of OX40 during primary P. yoelii infection (Zander et al., 2015), we further tested whether agonizing the OX40 co-stimulatory molecule during acute experimental malaria (Fig. 1A) would promote the accumulation and long-term maintenance of Plasmodium-specific memory CD4 T cells. Endogenous GP61–80-specific and total/bulk Py-GP infection-induced splenic CD4 T cell responses (CD4+CD44hi) were quantified at effector (days 7 and 14 p.i.) and memory (days 45, 60, and 90 p.i.) time points. As we reported (Zander et al., 2015), stimulating OX40 during primary P. yoelii infection decreased peak parasitemia and accelerated Plasmodium parasite clearance (Fig. 1B). Anti-OX40 treatment also increased the proportion and total number of GP61–80-specific memory CD4 T cells by 2 to 3-fold (Fig. 1C–E). Of note, α-OX40 treatment shifted the peak of the effector CD4 T cell proliferative expansion from day 7 to day 14 p.i. (Fig. 1E). Consistent with the GP61–80-specific memory CD4 T cell responses, the frequency (Fig. S1D) and total number (Fig. S1E) of total Py-GP infection-induced memory CD4+ T cell populations were increased ~2-fold following α-OX40 treatment. As inflammatory cytokines can erode the quantity and function of memory T cell populations (Harty and Badovinac, 2008), we investigated whether the observed elevation in memory CD4 T cells following OX40 ligation was associated with reduced inflammation as a consequence of the truncated infection. However, OX40 ligation triggered elevated inflammatory reactions that were sustained through day 12 p.i. (Fig. S1F–I), despite the 2-fold reduction in parasite burden at this time point (Fig. 1B). These data demonstrate that accelerated clearance of blood-stage Plasmodium parasites induced by agonizing the OX40 co-stimulatory receptor is associated with an enhanced accumulation of parasite-specific memory CD4 T cells.

Figure 1. Therapeutic ligation of OX40 during acute P. yoelii infection increases the magnitude of the P. yoelii GP61–80-specific CD4 T cell response.

Figure 1

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(A) Experimental design. Mice were infected with 1×106 transgenic Py-GP and administered either control rIgG or agonistic α-OX40 antibodies on days 7 and 10 p.i.

(B) Parasite growth and clearance kinetics.

(C) Proportions of tetramer+ GP61–80-specific splenic CD4+ T cells on day 45 p.i. in Py-GP-infected rIgG- and α-OX40-treated mice.

(D,E) Kinetics of the proportion (D) and total number (E) of GP61–80-specific CD4+ T cells.

Data (Mean +/− SEM) in (B,D,E) are pooled from 2–3 independent experiments per time point with 3–5 mice/group per experiment and were analyzed using multiple Student’s t tests while correcting for multiple comparisons using the Holm-Sidak method. *p<0.05 **p<0.01 ***p<0.0001. See also Figure S1

OX40 ligation during acute P. yoelii infection boosts the number of Ly6C+T-bet+ polyfunctional, Th1-like memory CD4 T cells

Both Th1 and Tfh cells contribute to Plasmodium parasite clearance (Langhorne et al., 1990, Perez-Mazliah and Langhorne, 2014, Perez-Mazliah et al., 2015, Spence and Langhorne, 2012, Stephens and Langhorne, 2010, Opata et al., 2015, Stephens et al., 2005, Stephens and Langhorne, 2006). Leishmania-specific Th1-like (Ly6C+T-bet+) effector CD4 T cells mediate enhanced parasite control (Peters et al., 2014) and we previously showed that therapeutic ligation of OX40 during primary P. yoelii infection expands effector Th1 cells (Zander et al., 2015). Thus, we next addressed whether agonistic α-OX40 treatment during an established Plasmodium infection affects the magnitude of Ly6C+ T-bet+ parasite-specific effector and memory CD4 T cell responses. By day 14 p.i., both proportions and total numbers of Ly6C+ Th1-like GP61–80-specific CD4 T cells were elevated 2 to 3-fold in α-OX40-treated mice, compared to rIgG-treated mice (Fig. 2A–C). Whereas Ly6C+ GP61–80-specific CD4 T cells in rIgG-treated mice gradually diminished over time, ligation of OX40 sustained the Ly6C+ GP61–80-specific CD4 T cell response through day 90 p.i. (Fig. 2B), which equated to a 7-fold increase in the total number of Ly6C+ GP61–80-specific memory CD4 T cells (Fig. 2C). The proportion of T-bet+ cells (Fig. 2A, right panels) and per cell expression of T-bet (gMFI, Fig. S2A) were highest among the Ly6C+ GP61–80-specific CD4 T cell subset. Consistent with elevated Ly6C+ Th1-like CD4 T cell numbers, we further identified increases in both the proportion (Fig. S2B) and total number (Fig. 2D) of T-bet+ GP61–80-specific CD4 T cells in α-OX40-treated mice, compared to rIgG-treated mice. Similar to NKT and anti-viral CD4 T cells (Marshall et al., 2011, Matsuda et al., 2006), T-bet is necessary (Fig. S2C,D), but not sufficient (e.g. Fig. 2F), for Ly6C expression on Plasmodium infection-induced CD4 T cells. Together, these data support that ligation of OX40 during primary P. yoelii infection promotes a 7-fold greater accumulation of T-bet-dependent Ly6C+ Th1-like memory CD4 T cells that persist for at least 90 days p.i.

Figure 2. Therapeutic ligation of OX40 during primary P. yoelii infection induces T-bet expression and expands polyfunctional Ly6C+ Th1-like memory CD4 T cell populations.

Figure 2

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(A–D). Mice were infected with 1×106 transgenic Py-GP and administered either control rIgG or agonistic α-OX40 antibodies on days 7 and 10 p.i.

(A) Proportions of Ly6C+ GP61–80-specific CD4 T cells and their expression of T-bet relative to Ly6Clo GP61–80-specific CD4 T cells on d14 p.i.

(B–D) Kinetics of the proportion (B) and total number (C) of Ly6C+ GP61–80-specific CD4 T cells and total number of T-bet+ (D) GP61–80 -specific CD4 T cells in rIgG- and α-OX40-treated mice.

(E) Experimental design. 10,000 naïve CD90.1+ SMARTA CD4 T cells were transferred to naïve CD90.2+ mice. Recipients were infected with Py-GP and treated with either agonistic α-OX40 or control IgG antibodies on d7 and 10 p.i.

(F) Proportions of Ly6C+T-bet+ memory SMARTA cells on day 45 p.i.

(G) Proportions of memory SMARTA cells expressing IFN-γ, TNF, and IL-2 on day 45 p.i.

(H) Summary of cytokine profiles of memory SMARTA cells from rIgG- and α-OX40-treated mice on day 45 p.i.

(I–L) Expression of Ly6C (I), IFN-γ (I,L), and IL-2/TNF (K) by total polyclonal (CD44hi) memory CD4 T cells on day 45 p.i.

Data (Mean +/− SEM) in (B–D) are pooled from 2–3 independent experiments per time point with 3–5 mice/group per experiment and were analyzed using multiple Student’s t tests while correcting for multiple comparisons using the Holm-Sidak method. Data (Mean +/− SD) in (H–L) derive from 4–5 mice/group and are representative of two independent experiments. *p<0.05 **p<0.01 N.S. = not significant. See also Figure S2

Expression of Ly6C is associated with enhanced effector cytokine production among effector and memory CD4 T cells (Hu et al., 2015, Marshall et al., 2011). Thus, we next tested whether ligation of OX40 during the primary P. yoelii infection also impacted the capacity of parasite-specific memory CD4 T cells to express pro-inflammatory cytokines. To do this, we used CD4+ T cell receptor transgenic cells specific for the LCMV GP61–80 epitope (SMARTA cells (Oxenius et al., 1998)) (Fig. 2E). Forty-five days p.i., we isolated and stimulated donor-derived, memory SMARTA cells ex vivo and performed intracellular cytokine staining for IFN-γ, TNF and IL-2. Agonistic α-OX40-treatment doubled the number of T-bet+Ly6C+ SMARTA cells (Fig. 2F) and the proportion of SMARTA cells competent to express IFN-γ, TNF and IL-2 (Fig. 2G,H). We further verified that total polyclonal (CD11ahiCD44hi) Py-GP infection-induced Ly6C+ memory CD4 T cells recovered from both control rIgG- and α-OX40-treated mice also exhibited a greater capacity to produce IFN-γ or TNF and IL-2, compared to Ly6Clo cells. Elevated cytokine expression (Fig. 2I–K) and the per cell expression of IFN-γ (Fig. 2L) were 30–40% greater among CD4 T cells recovered from α-OX40-treated mice, compared to cells recovered from rIgG-treated mice, which was sustained for at least 2 months after parasite clearance (Fig. S2E). Thus, compared to Ly6Clo CD4 T cells, parasite-specific Ly6C+ memory CD4 T cells exhibit enhanced polyfunctional cytokine expression and therapeutic ligation of OX40 during acute P. yoelii infection promotes the accumulation and maintenance of polyfunctional Th1-like Ly6C+T-bet+ memory CD4 T cells.

Ligation of OX40 during acute P. yoelii infection increases the number of parasite-specific CXCR5+ Tfh-like memory CD4 T cells that express Bcl-6 during recall responses

CXCR5+PD-1+Bcl-6+ Tfh cells are expanded during acute malaria and are critical for the secretion of isotype switched IgG and enhanced parasite control (Perez-Mazliah et al., 2015, Butler et al., 2012). Thus, we tested whether Plasmodium-specific Tfh cells have the capacity to form memory and persist. CXCR5 is the most reliable cell surface marker to track memory Tfh cells (Hale and Ahmed, 2015, Hale et al., 2013, Choi et al., 2013), as both PD-1 and Bcl-6 are down-regulated shortly after pathogen clearance (Pepper et al., 2011, Hale et al., 2013). Similarly, PD-1 and Bcl-6 also declined over the course of 60 days after Plasmodium infection (Fig. S3A–C). By contrast, the proportion of Tfh-like CXCR5+ GP61–80-specific CD4 T cells remained relatively stable and comprised ~20% of both effector (day 14 p.i.) and memory (>day 45 p.i.) populations (Fig. 3A,B). Ligation of OX40 during acute Plasmodium infection resulted in a 2–3-fold increase in the absolute number of CXCR5+ GP61–80-specific memory CD4 T cells that persisted through day 90 p.i. (Fig. 3C). At both effector and memory time points, the majority (85–95%) of Tfh-like CXCR5+ CD4 T cells are Ly6CloT-bet− Bcl-6+ (Fig. S3D–G) and Th1-like Ly6C+ cells are uniformly T-bet+CXCR5−Bcl-6− (Fig. S3E–G). Notably, substantial proportions of both effector and memory Ly6C+ Th1-like and CXCR5+Ly6Clo Tfh-like CD4 T cells express CXCR3 (Fig. S3H). These data support that the CXCR5−Ly6C+ and CXCR5+Ly6Clo phenotypes can be used to distinguish Th1- and Tfh-like Plasmodium-specific memory CD4 T cells, respectively. Moreover, CXCR3 expression may be a common signature of both Plasmodium-specific memory Th1 and Tfh cells, which is consistent with the identification of CXCR3+ effector Tfh cells in P. falciparum infected children (Obeng-Adjei et al., 2015).

Figure 3. Therapeutic ligation of OX40 during acute Plasmodium infection expands GP61–80-specific CXCR5+memory CD4 T cells that express Bcl-6 during recall.

Figure 3

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(A–C) Mice were infected with 1×106 transgenic Py-GP and administered either control rIgG or agonistic α-OX40 antibodies on days 7 and 10 p.i. Proportions of CXCR5+PD-1intBcl-6+ memory CD4 T cell on day 60 p.i. (A). Kinetics of the proportion (B) and total number (C) of CXCR5+PD-1int memory CD4 T cells.

(D,E) Py-GP-immune mice were challenged with 5×106 Py-GP parasites on day 60 p.i. (D) Representative flow plots showing the proportion of CXCR5+PD-1int GP61–80 -specific CD4 Tfh cells and their expression of Bcl-6. (E) Summary data showing the relative amount (gMFI) of Bcl-6 in GP61–80-specific secondary effector CD4 T cells 6 days after challenge of Py-GP-immune mice.

(F) CD44hiCXCR5+PD-1int memory CD4 T cells from rIgG- and α-OX40-treated mice were co-cultured with naïve B cells in the presence of α-CD3ε and α-IgM. Supernatants were assayed for secreted IgG on day 7.

(G) Py-GP-immune mice were challenged as in D and Hep17-specific serum IgG was measured before and 6 days later.

(H–J) Py-GP-immune mice harboring memory CD90.1+ SMARTA cells were challenged with 2×106 p.f.u. of LCMV. Frequency (H), total number (I) and expression of Bcl-6 (J) were evaluated in secondary effector SMARTA cells. Data (Mean +/− SEM) in (B,C,E) are pooled from 2–3 independent experiments per time point with 3–5 mice/group per experiment and were analyzed using Student’s t tests while correcting for multiple comparisons using the Holm-Sidak method. Data (Mean +/− SD) in (F) derive from 4 replicates, represent 2 independent experiments, and were analyzed by Student’s t-test. *p<0.05 **p<0.01 ***p<0.0001. See also Figure S3

Ligation of OX40 during acute Plasmodium infection did not differentially modulate Bcl-6 expression in CXCR5+ GP61–80-specific memory CD4 T cells through day 60 p.i. (Fig. S3A,B). However, compared to rIgG-treated mice, high-dose Plasmodium re-challenge of α-OX40-treated immune mice (60 days after primary infection) markedly increased the proportion of CXCR5+PD-1intBcl-6+ parasite-specific secondary effector CD4 T cells (Fig. 3D,E). Consistent with this enhanced Tfh-like phenotype after recall, naive (IgD+IgM+) B cells were stimulated to isotype switch and secrete 5 to 6-fold greater amounts of IgG when co-cultured with CXCR5+PD-1int memory Tfh cells sort purified from α-OX40-treated mice, compared to memory Tfh cells purified from rIgG-treated donor mice (Fig. 3F). Regarding the epitope-tagged Hep17 protein, by day 60 p.i. we observed a 2-fold increase in Hep17-specific IgG titers in α-OX40-treated mice, compared to rIgG-treated mice (Fig. 3G). Furthermore, homologous high-dose challenge with Py-GP lead to a 4-fold increase in titers in the α-OX40-treated mice, compared to a 2-fold increase in rIgG-treated mice (Fig. 3G).

We further hypothesized that Plasmodium infection-induced GP61–80-specific memory CD4 T cells expanded by α-OX40-treatment would also exhibit enhanced Tfh responses after heterologous infection. To test this, LCMV clone 13 was used to challenge rIgG- and α-OX40-treated, Py-GP-immune mice harboring GP61–80-specific SMARTA TCR Tg memory CD4 T cells. Strikingly, numbers of CXCR5+Bcl-6+ secondary effector Tfh cells were elevated ~2-fold (Fig. 3H,I) and relative expression of Bcl-6 increased by >20% (Fig. 3J) in cells recovered from mice treated with α-OX40 during acute Plasmodium infection. The expansion of Tfh cells was further associated with 2-fold larger GC B cell responses in LCMV-challenged mice (Fig. S3I,J). These data show that parasite-specific Ly6CloCXCR5+ Tfh-like memory CD4 T cells persist at least 90 days following experimental malaria and that therapeutic ligation of OX40 during acute Plasmodium infection enhances the recall and function of parasite-specific memory Tfh-like CD4 T cells after either homologous or heterologous boosting.

Ligation of OX40 during acute P. yoelii infection enhances the protective capacity of Plasmodium-specific memory CD4 T cells

To test the protective capacity of α-OX40-induced memory CD4 T cells, we sort-purified bulk memory CD4 T populations from rIgG- or α-OX40-treated mice and transferred 2×105 cells to T cell-deficient (Tcrα−/−) mice (Fig. 4A). Tcrα−/− recipients of memory CD4 T cells derived from either rIgG- or α-OX40 donors exhibited enhanced control of parasite replication, compared to mock-transfer Tcrα−/− mice (Fig. 4B). However, adoptive transfer of α-OX40 donor-derived memory CD4 T cells suppressed parasite growth by >2-fold compared to transfer of memory CD4 T cells derived from rIgG-treated donors (Fig. 4B,C). Suppression of parasite growth was associated with elevated IFN-γ expression by Ly6C+ secondary effectors (Fig. 4D,E). However, we also observed a 40–50% increase in the expansion of CXCR5+PD-1+Bcl-6+ secondary effector Tfh-like cells derived from α-OX40-treated memory CD4 T cells (Fig. 4F,G). Accordingly, merozoite surface protein (MSP)119-specific total IgG titers were elevated by >70% in Tcrα−/− mice seeded with memory CD4 T cells from α-OX40-treated donors (Fig. 4H). We repeated bulk transfer of memory CD4 T cells using recipient mice that lack both B and T cells (Rag1−/−). Donor-derived memory CD4 T cells engrafted and exhibited a CD62LloCD127loCD69hi effector phenotype in infected Rag1−/− recipients (Fig. S4A,B), yet we observed no differences in parasite control whether recipients were seeded with memory cells isolated from either rIgG- or α-OX40-treated donor mice (Fig. S4C). Furthermore, Rag1−/− mice from both experimental groups succumbed to P. yoelii infection between days 10 and 11 p.i. (Fig. S4D), supporting that B cells are essential for memory CD4 T cell-mediated protection following transfer. These results are consistent with previous studies showing that B cells are necessary for rescue of Plasmodium-infected Rag2−/− mice following CD4 T cell transfer (Stephens et al., 2005, Stephens and Langhorne, 2010). These data show that stimulating OX40 during a Plasmodium-specific primary effector CD4 T cell response boosts the protective capacity of memory populations, which is associated with amplified Tfh-like recall and enhanced B cell helper functions.

Figure 4. Therapeutic ligation of OX40 during acute Plasmodium infection enhanced both Th1- and Tfh-like recall responses and per cell protective capacity of memory CD4 T cells.

Figure 4

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(A) Experimental design. Mice were infected with 1×106 Py and administered either control rIgG or α-OX40 antibodies on day 7 and 10 p.i. On 75 day p.i., memory CD4 T cells were sorted and transferred (2×105) into groups of Tcrα−/− mice. Recipients were challenged with P. yoelii and parasite growth kinetics were measured. Cells were analyzed on day 14 p.i. Statistical analyses (*p<0.05) reflect comparisons between recipients of memory cells from rIgG- and α-OX40-treated donor mice.

(B,C) Kinetics of parasite growth (B) and summary data (C) showing parasitemia on day 14 p.i.

(D–F) Proportions of donor-derived CD4 T cells expressing either Ly6C (D), IFN-γ (D,E), or CXCR5+PD1+ Tfh cells expressing Bcl-6 (F).

(G) Total number of CXCR5+PD1+Bcl-6+ Tfh cells recovered from recipients.

(H) MSP119-specific serum antibody titers in mice seeded with memory CD4 T cells derived from rIgG- and α-OX40-treated donors.

Data (Mean +/− SEM) in (B,C,E,G,H) are pooled from 2 independent experiments with 3–4 mice/group per experiment and were analyzed using either one-way ANOVA (B,C) or Student’s t tests (E,G,H). *p<0.05 **p<0.01. N.S. = not significant. See also Figure S4.

Plasmodium-specific, Th1-like Ly6C+ memory CD4 T cells exhibit Tfh function during recall responses

We observed both Th1- and Tfh-like activity following the transfer and recall of bulk memory CD4 T cells, suggesting that either substantial heterogeneity or functional plasticity may exist within the Plasmodium-specific memory CD4 T cell compartment. To evaluate the phenotypic stability and activity of Th1- and Tfh-like memory CD4 T cells during recall and determine if B cell helper function is restricted to the Tfh-like populations, performed adoptive transfer studies (Fig. 5A). To simplify sort-purification, we enriched Th1- and Tfh-like memory CD4 T cell populations based on differential expression of Ly6C, given that the development of Th1-like Ly6C+ memory CD4 T cells is T-bet-dependent (Fig. S2C,D and (Marshall et al., 2011, Matsuda et al., 2006)) and Tfh-like CXCR5+ Bcl-6+ memory CD4 T cells are only found within the Ly6Clo CD4 T cell population (Fig. S3D,E and S5A,B). Following their adoptive transfer and recall, Plasmodium-specific Ly6C+ memory CD4 T cells underwent secondary expansions that were numerically equivalent to Ly6Clo memory CD4 T cells (Fig. 5B,C and Fig. S5C), which was unexpected given reported proliferative deficits among Ly6C+ CD4 T cells (Hu et al., 2015, Marshall et al., 2011). Moreover, memory CD4 T cell populations derived from α-OX40-treated mice expanded ~4-fold more than memory CD4 T cells derived from control rIgG-treated donor mice, independent of Ly6C expression (Fig. 5C and Fig. S5C). Approximately 50% of secondary effectors expressed Ly6C following transfer and recall of Ly6Clo memory CD4 T cells (Fig. 5D). By contrast, ~25–35% of donor Ly6C+ memory CD4 T cells down-regulated Ly6C following transfer and recall (Fig. 5D and Fig. S5D), supporting that Plasmodium-specific Ly6C+ memory CD4 T cells retain the capacity to proliferate and exhibit a high degree of phenotypic inter-conversion between Ly6C+ and Ly6Clo memory CD4 T cells during recall in response to homologous infection. As expected, Ly6C+ secondary effector progeny of either Th1- or Tfh-like memory cells expressed more T-bet relative to their Ly6Clo counterparts (Fig. S5E). However, despite this apparent Th1-like bias, more than twice as many Ly6C+ secondary effectors co-expressed CXCR5 and Bcl-6, compared to Ly6Clo effector CD4 T cells (Fig. 5D,E and Fig. S5F). Thus, Ly6C+ secondary effectors derived from either Ly6C+ or Ly6Clo memory CD4 T cells appeared to more efficiently recall or acquire a Tfh-like phenotype during recall responses, suggesting that Tfh activity may reside within the Ly6C+ pool of secondary effectors.

Figure 5. Both Ly6Clo and Ly6C+ Plasmodium-specific memory CD4 T cells can give rise to Tfh-like secondary effectors.

Figure 5

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(A) Experimental design. CD45.1+ mice were infected with 1×106 Py and administered either control rIgG or α-OX40 on days 7 and 10 p.i. On day 48 p.i., 2×105 total (CD11ahiCD44hi) infection-induced Ly6C+ memory CD4+ T cells were sorted and transferred into congenic CD45.2+ mice. Recipient mice were infected with Py one day later. CD4 T cell responses were assessed on d7 p.i.

(B,C) Representative flow plots (B) and summary graph (C) showing expansion of CD45.1+ cells on day 7 p.i.

(D) Phenotype and CXCR5 and Bcl-6 expression among recovered CD45.1+ Ly6Clo and Ly6C+ secondary effector CD4 T cells.

(E) Summary data showing the proportion of Ly6Clo and Ly6C+ CD45.1+ secondary effector cells expressing CXCR5 and Bcl-6.

(F) Summary data of Bcl-6 expression (gMFI) in rIgG- and α-OX40-treated donor-derived CD45.1+ secondary effector CD4 T cells.

Data (Mean +/− SD) in (C) were analyzed using one-way ANOVA. Data (Mean +/− SEM) in (E–F) are pooled from 2 independent experiments with 5 mice/group and were analyzed using Student’s t tests. *p<0.05 **p<0.01 ***p<0.0001. See also Figure S5

Regarding the impact of α-OX40 treatment, both the proportion of CXCR5+Bcl-6+ Tfh cells (Fig. 5E and Fig. S5F) and the amount of Bcl-6 (Fig. 5F) were elevated ~25% in Ly6C+ secondary effectors derived from α-OX40-treated mice, compared to those derived from IgG-treated mice. Identical patterns were observed following recall of Py-GP GP61–80-specific memory CD4 T cells (Fig. S5G). Thus, both Ly6Clo and Ly6C+ Plasmodium-specific memory CD4 T cells are endowed with the capacity to give rise to Tfh-like secondary effectors. Collectively, our data support that phenotypically distinct Plasmodium-specific Th1- and Tfh-like memory CD4 T cell subsets display functional overlap during secondary recall responses. Moreover, our data show that Tfh-like qualities can be recalled from Ly6C+ Th1-like memory CD4 T cells, features that are enhanced by therapeutic ligation of OX40.

Parasite-specific, Th1-like Ly6C+ memory CD4 T cells exhibit potent B cell helper function

To directly compare the capacity of Ly6C+ Th1- and Ly6Clo Tfh-like memory CD4 T cells to support B cell helper function, and to determine whether OX40 stimulation imprinted on this function, we performed additional in vivo and in vitro B cell differentiation and activation studies (Sage and Sharpe, 2015). Strikingly, transfer of Th1-like Ly6C+ memory CD4 T cells stimulated elevated titers of parasite-specific IgG2b in recipient mice, compared to transfer of Tfh-like Ly6Clo memory CD4 T cells (Fig. 6A). Consistent with these in vivo data, Th1-like Ly6C+ memory CD4 T cells sort-purified from either rIgG- or α-OX40-treated mice exhibited superior B cell helper function, compared to their Ly6Clo counterparts (Fig. 6B). Of note, B cells cultured with Ly6C+ memory CD4 T cells sorted from α-OX40-treated mice secreted more IgG, compared to B cells cultured with Ly6C+ memory CD4 T cells sort-purified from rIgG-treated mice (Fig. 6B). In line with these in vitro studies, transfer of Ly6C+ memory CD4 T cells isolated from α-OX40-treated mice more effectively limited parasite replication in T cell-deficient recipients following P. yoelii challenge (Fig. 6C), which was associated with >50% increases in T cell-dependent germinal center B cell class-switching (Fig. 6D,E) and circulating titers of MSP119-specific IgG (Fig. 6F). Thus, Plasmodium-specific Ly6C+ Th1-like memory CD4 T cells exhibit significant functional heterogeneity and are capable of providing robust B cell help during recall responses. Collectively, our data provide both in vivo and in vitro evidence that Plasmodium-specific Th1-like Ly6C+T-bet+ memory CD4 T cells more effectively support secondary humoral immune reactions compared to Ly6Clo populations in which CXCR5+Bcl-6+ Tfh-like cells reside. Moreover, our data demonstrate that the host-specific co-stimulatory molecule OX40 can be therapeutically targeted during malaria to enhance the B cell helper function and protective capacity of polyfunctional Ly6C+ Th1-like memory CD4 T cells.

Figure 6. Ligation of OX40 during an acute P. yoelii infection bolsters the anti-parasitic and B cell helper capacity of Plasmodium-specific, Th1-like Ly6C+ memory CD4 T cells.

Figure 6

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(A–F) Mice were infected with 1×106 Py and administered either control rIgG or agonistic α-OX40 antibodies on days 7 and 10 p.i.

(A) MSP119-specific serum IgG2b titers in Plasmodium-infected mice seeded with either Ly6Clo or Ly6Chi memory CD4 T cells.

(B) On day 50 p.i., Ly6C+ and Ly6Clo memory CD4 T cells were sort-purified from rIgG- and α-OX40-treated mice and co-cultured with naïve (CD19+IgDhi) B cells in the presence of α-CD3ε and α-IgM. Secreted IgG was measured on day 5.

(C) Ly6C+ and Ly6Clo memory CD4 T cells from rIgG- and α-OX40 treated-mice were sort-purified and transferred (2×105) into Tcrα−/− mice. Recipients were infected the following day. Parasite burdens on day 16 p.i. are shown.

(D,E) Proportion (D) and summary (E) of class-switched (IgDloIgMlo) germinal center B cells (GL-7+CD95+) in Tcrα−/− mice seeded with Th1-like Ly6C+ memory CD4 T cells derived from either rIgG- or α-OX40-treated donors.

(F) MSP119-specific serum IgG titers in memory CD4 T cell recipient Tcrα−/− mice on day 16 p.i.

Data (Mean +/− SD) in (A–F) are from 5 mice/group, represent 2–3 independent experiments, and were analyzed by either Student’s t tests (A,B, D–F) or one-way ANOVA (C). *p<0.05 **p<0.01. N.S. = not significant.

Discussion

Multiple studies show that OX40 signaling is important for promoting effector CD4 T cell proliferation and survival (Murata et al., 2000, Kopf et al., 1999, Soroosh et al., 2007, Gramaglia et al., 2000, Song et al., 2008, Song et al., 2007, Song et al., 2005). Our data show that OX40 ligation during established malaria can also enhance the function of memory CD4 T cells. OX40 signaling is known to activate the PI3K/Akt (Song et al., 2004), NfKB (Song et al., 2008), NFAT (So et al., 2006), and mTOR (Song et al., 2004) pathways, as well as regulate the activity of c-Myc (Haque et al., 2016). Each of these circuits modulates memory T cell formation (Angelosanto and Wherry, 2010, Hale et al., 2013, Youngblood et al., 2013a, Youngblood et al., 2013b). Understanding the mechanisms by which OX40, alone or in combination with other co-stimulatory (Wikenheiser et al., 2016) or co-inhibitory (Wykes and Lewin, 2017) pathways, impacts memory CD4 T cell function may be key for developing strategies to enhance protection against chronic infections or cancer.

The origins, phenotype, and function of memory CD4 T cells and their capacity for protection during recall remain enigmatic (Opata and Stephens, 2013, Seder et al., 2008). Following acute bacterial infection, CXCR5+PD-1int CD4 T cells exhibiting characteristics of either T central memory or Tfh cells have been identified (Pepper et al., 2011). Compared to other T cell subsets, including Th1-like Ly6C+T-bet+ CD4 T cells, Tfh-like CXCR5+Ly6CloT-betlo memory cells induced by acute viral infection exhibit enhanced longevity, elevated per cell proliferative capacity, and enhanced B cell helper function following their adoptive transfer and recall (Marshall et al., 2011, Hale et al., 2013). Following Leishmania infection, CXCR3+Ly6C+T-bethi effector CD4 T cells are defined as short-lived Th1 cells that mediate enhanced parasite control, yet exhibit limited capacity to form memory or undergo secondary expansion, compared to their less-differentiated CXCR3loLy6CloT-betlo counterparts (Peters et al., 2014). Here evidence is presented supporting that Plasmodium-specific Th1-like Ly6C+T-bet+ memory CD4 T cells exhibit equivalent or enhanced proliferative potential, CXCR5 and Bcl-6 expression, and B cell helper function during recall, compared to pools of Ly6Clo populations that harbor CXCR5+ memory Tfh cells. The proliferative potential of Plasmodium-specific Ly6C+ Th1-like memory CD4 T cells contrasts sharply with the reported inability of virus- and Leishmania-specific Ly6C+ Th1-like effector and memory CD4 T cells to proliferate (Hu et al., 2015, Marshall et al., 2011). The basis for the significant functional plasticity among Plasmodium-specific memory CD4 T cells may be linked to the distinct inflammatory environments and antigen presenting cell types expanded during malaria. Direct comparative studies will be required to delineate the molecular and transcriptional features of Plasmodium-specific Th1- and Tfh-like memory CD4 T cells that distinctly govern their development, plasticity, and protective function.

An emerging literature supports that anti-microbial effector CD4 T cells can exhibit mixed Th1- and Tfh-like phenotype and function (Carpio et al., 2015, Li et al., 2016, Nakayamada et al., 2011, Obeng-Adjei et al., 2015, Velu et al., 2016, Wikenheiser et al., 2016). Viral infections are linked to expansion of effector Tfh cells that express CXCR3 and co-produce IFN-γ and IL-21 following experimental SIV infection (Velu et al., 2016). IFN-γ+IL-21+ICOS+Bcl-6+ Mycobacterium tuberculosis-specific effector CD4 T cell populations have been identified (Li et al., 2016) and Toxoplasma gondii-specific effector CD4 T cells are reported to adopt a transitional stage characterized by co-expression of IL-21, Bcl-6, and T-bet (Nakayamada et al., 2011). Recent studies also identify Tfh cells exhibiting characteristics of Th17, Th1, and Th2 effector CD4 T cells in vivo (Morita et al., 2011, Obeng-Adjei et al., 2015) and in vitro experiments show that Th17-like effector Tfh cells exert potent B cell helper function, relative to Th1- and Th2-like effector Tfh cells (Lu et al., 2011, Mitsdoerffer et al., 2010). During chronic experimental malaria, CD4 T cells can co-express CXCR5, IL-21, IL-10 and IFN-γ (Carpio et al., 2015, Perez-Mazliah et al., 2015). Despite the consistent reports of expanded populations of effector Th1/Tfh-like CD4 T cells following microbial infection, whether these cells represent either distinct CD4 T cell subsets or a continuum of phenotype and function, and whether they retain capacity to form stable memory populations that distinctly mediate protective immunity have not been thoroughly addressed. Our data support that Plasmodium-specific Th1-like effector and memory cells are transcriptionally distinct from Tfh-like cells, which is consistent with recent data from others (Lonnberg et al., 2017, Hale et al., 2013). However, Plasmodium-specific Th1- and Tfh-like effector CD4 T cells exhibit phenotypic similarities, including expression of CXCR3. Our data show that this phenotype is stable through memory formation. Our data also show that Th1- and Tfh-like memory CD4 T cell subsets display functional overlap during recall responses, with both Th1- and Tfh-like memory subsets giving rise to secondary effectors that robustly proliferate and exhibit phenotypic and functional features of Tfh cells. Thus, substantial proliferative capacity and potent B cell helper activity unexpectedly resides within the Plasmodium-specific Ly6C+ Th1-like memory CD4 T cell pool.

Impaired humoral immunity during malaria is linked to expansions of circulating Th1-like effector Tfh cells (reviewed in (Hansen et al., 2016)). The acquisition of Th1-associated CXCR3, T-bet, and IFN-γ expression by pre-Tfh cells is reported to constrain their migration and development into competent B helper cells in follicles (Ioannidis et al., 2016, Ryg-Cornejo et al., 2016). We and others have shown that excessive IFN-γ limits humoral immunity (Zander et al., 2015, Ryg-Cornejo et al., 2016). In line with a potential deleterious effect of Tfh-derived IFN-γ expression, we recently identified that B cell-intrinsic IFN-γ signaling limits Plasmodium-specific GC B cell reactions (Guthmiller et al., 2016). Despite these analyses of effector Tfh responses, our current experiments show that potent B cell helper activity can be recalled from Th1-like, CXCR3+Ly6C+T-bet+ memory CD4 T cells, which can express IFN-γ ex vivo. However, whether Th1-like Tfh memory cells either express IFN-γ in vivo or whether IL-21 (Perez-Mazliah et al., 2015) or IL-10 (Guthmiller et al., 2016) counteract the activity of IFN-γ during recall responses remains unknown. Although IL-21 expression was undetectable in Tfh- and Th1-like memory CD4 T cells ex vivo (not shown), current work in our laboratory is focused on dissecting these functional parameters as well as the transcriptional networks regulating Plasmodium-specific effector and memory Tfh cell development in vivo. Such information will be critical for developing new strategies to enhance the formation and function of long-lived, Plasmodium-specific memory B cells and plasma cells and durable protection against malaria.

Experimental Procedures

Mice, parasites, viruses, and biologics

Male and female C57BL/6 wild type, Tbx21−/−, Rag1−/− and Tcrα−/− mice (6–8 weeks, 16–21g) were purchased from Jackson Laboratories. SMARTA mice were generously provided by Dorian McGavern (Scripps) via Allan Zajac (UAB). The University of Oklahoma Health Sciences Center and University of Iowa Institutional Animal Care and Use Committees approved all experiments. Plasmodium yoelii (clone 17XNL, BEI Resources/MR4/ATCC) was inoculated into mice intravenously. To generate the T cell epitope tagged transgenic parasites (see Fig. S1 and Table S1), a dispensable genetic locus (“p230p”) was stably targeted with an expression cassette of Hep17 protein tagged with the GP61–80 CD4 T cell epitope (amino acids 61–80 from LCMV glycoprotein). Standard methods (Jongco et al., 2006, Lindner et al., 2013) were used to create and integrate a linearized pDEF construct, select transgenic parasites, and obtain two pure, clonal populations. Parasitemia was measured using flow cytometry as previously described (Malleret et al., 2011). LCMV clone 13 (2×106 plaque forming units) was administered i.p. 50 µg of rIgG or α-OX40 antibody (clone OX86, BioXCell) were administered i.p. on the days indicated in figure legends.

Flow cytometry

T cells were stained with H2-Kb/GP61–80 tetramers (NIH) and antibodies against mouse CD4 (clone GK1.5, Biolegend), CD11a (clone M17/4, eBioscience), CD25 (clone PC61, Biolegend), CD44 (clone IM7, Tonbo), Ly6C (clone HK1.4, Biolegend), CXCR3 (clone CXCR3-173, Biolegend), and PD-1 (clone RMP1-30, Biolegend). B cells were stained with CD19 (clone 6D5, Tonbo), T and B cell activation antigen (clone GL-7, BD Biosciences), CD95 (clone Jo2, BD Biosciences), IgD (clone 11-26c.2a, BD Biosciences), and IgM (clone RMM-1, eBioscience). Cells were stimulated with either GP61–80 peptide antigen or PMA and ionomycin in the presence of monensin, permeabilized with cytofix/cytoperm (BD Bioscience), and stained with anti-mouse IFN-γ (clone XMG1.2, eBioscience), anti-IL-2 (clone JES6-5H4, eBioscience), and anti-TNF (clone MP6-XT22, eBioscience). For Tfh analyses, cells were stained with anti-CXCR5 (clone 2G8, BD Biosciences) followed by staining with fluorochrome-conjugated anti-CD4, anti-CD44, anti-Ly6C, anti-CXCR3, and anti-PD-1. Anti-T-bet (clone 4B-10, Biolegend) and anti-Bcl-6 (clone K112-91, BD Biosciences) were applied after fixation and permeabilization of cells using FoxP3/Transcription Factor Staining Buffer Kits (Tonbo). Data were acquired using either Stratedigm S1200Ex or BD LSR II flow cytometers and analyzed using FlowJo software (Tree Star, Inc.).

Cell sorting and adoptive transfer

CD4 T cells were enriched (AutoMACS Pro-Separator, Miltenyi), stained for various markers (CD90, CD4, Ly6C, CXCR5, and/or PD-1), and sort-purified using either FACSJazz or Aria II instruments (BD Biosciences). Memory CD4 T cell subsets were adoptively transferred (2×105) via tail vein.

CD4 T cell-B cell co-cultures

Memory cells were sorted and co-cultured with naïve (CD19+IgDhiCD44lo) B cells (1:1) for 5–7 days in 96 well round-bottom plates containing 5 µg/ml of anti-CD3ε (clone 145-2C11) and 5 µg/ml anti-mouse IgM (Jackson ImmunoResearch) as described (Sage and Sharpe, 2015).

ELISA

Plates (Nunc) were coated with recombinant P. yoelii MSP119 (BEI Resources/MR4/ATCC) and blocked with 2.5% BSA + 5% normal goat serum. MSP119-specific total IgG or IgG2b was detected using HRP-conjugated goat anti-mouse-IgG (Jackson ImmunoResearch). Plates were developed with SureBlue Reserve TMB Kit (KPL) and analyzed with a Spectra Max 340 (Molecular Devices).

Statistical Analyses

Tests of statistical significance were conducted using Prism 6 software (GrapPad) and either non-parametric unpaired Student’s t-tests or one-way ANOVA as indicated in figure legends.

Supplementary Material

supplement

Highlights.

  • Reagents generated to study Plasmodium-specific memory CD4 T cell development

  • Th1- and Tfh-like memory CD4 T cells exhibit functional overlap during recall

  • Potent B cell helper function can be recalled from Th1-like memory cells

  • Functions of parasite-specific memory CD4 T cells are enhanced by ligation of OX40

Acknowledgments

We thank Drs. Martin Richer (McGill University) and Lauren Zenewicz (OUHSC) for critical feedback. This work was supported by grants from the National Institutes of Health/National Institute of Allergy and Infectious Diseases (T32AI007633 to R.A.Z.; K22AI101039 and R01AI123341 to S.E.L.; R01AI125446 and R01AI127481 to N.S.B.), the American Heart Association (16PRE27660002 to J.J.G.), and the National Institutes of Health/National Institute of General Medical Sciences under grant number 8P20GM103447.

Footnotes

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Author Contributions

RAZ designed, performed, analyzed, and interpreted experiments and wrote the paper. RV, ADP, JJG, and ACG performed, analyzed, and interpreted experiments. SEL and AMV assisted with the generation of the transgenic parasites. SHIK provided resources to generate the transgenic parasites. NSB conceived and generated transgenic parasites, designed, performed, analyzed, and interpreted experiments, and wrote the paper.

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