Bystander Activation and Anti-Tumor Effects of CD8+ T Cells Following Interleukin-2 Based Immunotherapy Is Independent of CD4+ T Cell Help

<div><p>We have previously demonstrated that immunotherapy combining agonistic anti-CD40 and IL-2 (IT) results in synergistic anti-tumor effects. IT induces expansion of highly cytolytic, antigen-independent “bystander-activated” (CD8⁺CD44^high) T cells displaying a CD25⁻NKG2D⁺ phenotype in a cytokine dependent manner, which were responsible for the anti-tumor effects. While much attention has focused on CD4+ T cell help for antigen-specific CD8+ T cell expansion, little is known regarding the role of CD4+ T cells in antigen-nonspecific bystander-memory CD8+ T cell expansion. Utilizing CD4 deficient mouse models, we observed a significant expansion of bystander-memory T cells following IT which was similar to the non-CD4 depleted mice. Expanded bystander-memory CD8+ T cells upregulated PD-1 in the absence of CD4+ T cells which has been published as a hallmark of exhaustion and dysfunction in helpless CD8+ T cells. Interestingly, compared to CD8+ T cells from CD4 replete hosts, these bystander expanded cells displayed comparable (or enhanced) cytokine production, lytic ability, and in vivo anti-tumor effects suggesting no functional impairment or exhaustion and were enriched in an effector phenotype. There was no acceleration of the post-IT contraction phase of the bystander memory CD8+ response in CD4-depleted mice. The response was independent of IL-21 signaling. These results suggest that, in contrast to antigen-specific CD8+ T cell expansion, CD4+ T cell help is not necessary for expansion and activation of antigen-nonspecific bystander-memory CD8+ T cells following IT, but may play a role in regulating conversion of these cells from a central memory to effector phenotype. Additionally, the expression of PD-1 in this model appears to be a marker of effector function and not exhaustion.</p></div

Anti-tumor effects of IT in CD4 knockout mice.

<p>3LL tumor bearing WT or CD4 knockout (B6.129S2-CD4^tm1Mak/J) mice were treated with IT or PBS/rIgG (control) and survival and tumor growth were measured. For <i>in vivo</i> tumor studies one million 3LL cells were administered by s.c. injection into the flank of C57BL/6 mice seven days prior to initiation of therapy. (<b>a</b>) Survival. (<b>b</b>) Mean tumor volume with SEM. (<b>c–f</b>) Growth plots of individual tumors in each group. N = 12 mice per group. (*<i>P</i><.05, **<i>P</i><.01, ***<i>P</i><.001).</p

Increased number of PD-1+ memory CD8+ T cells after IT in CD4+ T cell deficient mice.

<p>Control or CD4 deficient (depleted or knockout) C57BL/6 mice were treated with IT or PBS/rIgG (control) and PD-1 expression on memory T-cells was quantified by flow cytometric analysis 11 days after the initiation of IT. (<b>a</b>) Representative dot plots for PD-1+ gating on CD8+ CD44^high cells in the spleens of CD4+ depletion model mice. Number of PD-1+ memory (CD44^high) CD8+ T cells in spleens (<b>b,d</b>) and LNs+ (<b>c,e</b>) of IT or vehicle treated mice in CD4+ depletion (<b>b,c</b>) or CD4 knockout (<b>d,e</b>) models. Results are representative of two (CD4 knockout) or three (CD4 depletion) independent experiments with a minimum of three mice per group. (*<i>P</i><.05, **<i>P</i><.01, ***<i>P</i><.001).</p

Memory CD8+ T cell function after IT in CD4+ T cell deficient models.

<p>Control or CD4 deficient (depleted or knockout) C57BL/6 mice were treated with IT or PBS/rIgG (control) and assessed for function of memory CD8+ T cells 11 days after the initiation of IT. NKG2D and granzyme B expression were quantified by flow cytometric analysis. Interferon gamma production was quantified by flow cytometric analysis after <i>in vitro</i> restimulation of splenocytes with PMA/Ionomycin (0.16/1.6 ug/ml) for one hour followed by incubation with golgi stop (0.7 ug/ml) for three hours. CD8+ T cell killing function was assayed by scintillation counting using an <i>in vitro</i> redirected lysis assay with ⁵¹Cr labeled P815 target cells incubated for 30 minutes with 10 ug/mL anti-CD3e. (<b>a,f</b>) NKG2D expression on memory CD8+ T cells in CD4 depletion (<b>a</b>) and knockout (<b>f</b>) models. Representative dot plots for NKG2D+ CD25− gating are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102709#pone-0102709-g001" target="_blank">Figure 1</a>. (<b>b</b>) Representative dot plots for IFNγ+ gating on CD8+ CD44^high cells in the spleens of CD4+ depletion model mice. (<b>c,g</b>) Interferon gamma production by memory CD8+ T cells in CD4 depletion (<b>c</b>) and knockout (<b>g</b>) models. (<b>d</b>) Representative dot plots for Granzyme B+ gating on CD8+ CD44^high cells in the spleens of CD4+ depletion model mice. (<b>e,h</b>) Granzyme B expression by memory CD8+ T cells in CD4 depletion (<b>e</b>) and knockout (<b>h</b>) models. Killing function of splenocytes from CD4 depleted (<b>i</b>) or CD knockout (<b>j</b>) mice expressed as percentage of maximal lysis. Results are representative of two (CD4 knockout) or three (CD4 depletion) independent experiments with a minimum of three mice per group. (*<i>P</i><.05, **<i>P</i><.01, ***<i>P</i><.001).</p

CD40/IL-2 Immunotherapy induces massive expansion of bystander memory CD8+ cells and anti-tumor effects are CD8 dependent.

<p>Three C57BL/6 mice per group were treated with IT or PBS/rIgG (control) and effects on CD8+ T cell expansion were quantified by flow cytometric analysis 11 days after the initiation of therapy. For <i>in vivo</i> tumor studies one million 3LL cells were administered by s.c. injection into the flank of C57BL/6 mice seven days prior to initiation of therapy. Six to eight 3LL bearing mice were treated with IT and/or CD8+ T cell depletion to examine CD8+ dependence of anti-tumor effects. (<b>a</b>) Gating strategy for bystander memory CD8+ CD44^high NKG2D+ CD25− cells. (<b>b–e</b>) Expansion of bystander memory CD8+ T cells in the spleen and lymph nodes of IT or vehicle treated mice expressed as total numbers (<b>b,c</b>) or as a percentage of total CD8+ T cells (<b>d,e</b>). Effects of IT and/or CD8 depletion on tumor growth (<b>f</b>) and survival (<b>g</b>).</p

PD-1 expression on central and effector memory CD8+ T cells after IT in CD4+ T cell depleted mice.

<p>Control or CD4+ depleted C57BL/6 mice were treated with IT or PBS/rIgG (control). CD62L and PD-1 expression on memory T-cells was quantified by flow cytometric analysis 11 days after the initiation of IT. (<b>a,b</b>) Examples of the gating strategy for PD-1 expression on the CM and EM components of the memory CD8+ T cell compartment. The majority of cells in the memory compartment are CM in untreated mice (<b>a</b>) and EM in IT treated mice (<b>b</b>). (<b>c</b>) PD-1 expression on CM and EM cells. (<b>d</b>) The composition of the memory CD8+ T cell compartment in control or CD4 depleted mice treated with IT or PBS/rIgG. PD-1+ CM cells (<b>e</b>) and PD-1+ EM cells (<b>f</b>) as a percentage of the total memory CD8+ T cell compartment. Results are representative of two to three independent experiments with 3 mice per group (*<i>P</i><.05, **<i>P</i><.01, ***<i>P</i><.001, ****<i>P</i><.0001).</p

IT induced expansion of memory CD8+ T cells in CD4+ T cell deficient models.

<p>Control or CD4 deficient (depleted or knockout) C57BL/6 mice were treated with IT or PBS/rIgG (control) and effects on CD8+ T cell expansion were quantified by flow cytometric analysis 11 days after the initiation of IT. CD8+ (<b>a,b</b>) and memory CD8+ (<b>c,d</b>) T cell numbers in the LNs (<b>a,c</b>) and spleens (<b>b,d</b>) of control or CD4+ T cell depleted mice treated with vehicle or IT. (<b>e</b>) BrdU incorporation in CD8+ T cells from spleens of control or CD4+ T cell depleted mice treated with vehicle or IT. CD8+ (<b>f,g</b>) and memory CD8+ (<b>h,i</b>) T cell numbers in the LNs (<b>f,h</b>) and spleens (<b>g,i</b>) of wild-type or CD4 knockout mice treated with vehicle or IT. Results are representative of two (CD4 knockout) or three (CD4 depletion) independent experiments with a minimum of three mice per group. (*<i>P</i><.05, **<i>P</i><.01, ***<i>P</i><.001).</p