Adoptive immunotherapy of cancer with polyclonal, 108-fold hyperexpanded, CD4+ and CD8+ T cells

Abstract

T cell-mediated cancer immunotherapy is dose dependent and optimally requires participation of antigen-specific CD4+ and CD8+ T cells. Here, we isolated tumor-sensitized T cells and activated them in vitro using conditions that led to greater than 108-fold numerical hyperexpansion of either the CD4+ or CD8+ subset while retaining their capacity for in vivo therapeutic efficacy. Murine tumor-draining lymph node (TDLN) cells were segregated to purify the CD62Llow subset, or the CD4+ subset thereof. Cells were then propagated through multiple cycles of anti-CD3 activation with IL-2 + IL-7 for the CD8+ subset, or IL-7 + IL-23 for the CD4+ subset. A broad repertoire of TCR Vbeta families was maintained throughout hyperexpansion, which was similar to the starting population. Adoptive transfer of hyper-expanded CD8+ T cells eliminated established pulmonary metastases, in an immunologically specific fashion without the requirement for adjunct IL-2. Hyper-expanded CD4+ T cells cured established tumors in intracranial or subcutaneous sites that were not susceptible to CD8+ T cells alone. Because accessibility and antigen presentation within metastases varies according to anatomic site, maintenance of a broad repertoire of both CD4+ and CD8+ T effector cells will augment the overall systemic efficacy of adoptive immunotherapy.

Figure 1

Proliferation and efficacy of CD62L^low TDLN cells cultured with IL-2 +/- IL-7. (A) Freshly isolated whole TDLN cells were stained for expression of TCR and CD62L (left panel). The purified CD62L^low subset was stained for TCR and CD62L expression (center panel), or for CD4 and CD8 expression (right panel). (B) CD62L^low TDLN cells were activated with immobilized anti-CD3 mAb for 2 days then cultured in medium with IL-2 (4 U/ml) (closed circle) or the combination of IL-2 and IL-7 (10 ng/ml) (open circle). Cells density was adjusted to 10⁵/ml on days 5 and 9 and total proliferation was calculated. (C) Mice bearing 3-day s.c tumors were treated with 5 Gy TBI then received adoptive transfer of 5 × 10⁶ T cells cultured for 9 days with IL-2 alone (open circle), IL-2 + IL-7 (open triangle), or HBSS (closed circle). Each treatment group is significantly different than HBSS control (P < 0.01).

Figure 2

Restimulation of activated T cells induces additional proliferation with retention of specific anti-tumor efficacy. (A) CD62L^low TDLN cells were activated with anti-CD3 mAb from day 0–2 and again for 14 hrs on day 15. T cells were cultured in the presence of IL-2 (4 U/ml) (closed circle) or IL-2 plus IL-7 (10 ng/ml) (open circle) and the total proliferation is indicated. (B) FACS analysis of activated T cells on day 23 of culture stained for CD4 and CD8. (C) Mice bearing 10-day pulmonary metastases of either MCA205 or MCA207 tumors were pre-treated with 5 Gy TBI then received adoptive transfer of 2.5 × 10⁷ cells cultured with IL-2 alone, the combination of IL-2 plus IL-7, or control HBSS as indicated. Difference between the groups bearing MCA 205 treated with T cells cultured with IL-2 or IL-2 plus IL-7 and all other groups is (P < 0.01). (D) Mice bearing 3-day s.c tumors were pre-treated with 5Gy TBI followed by injection of; HBSS (closed circles), 5 × 10⁶ T cells activated for 5 days (open circles, P < 0.01), 5 × 10⁶ restimulated T cells at day 23 of culture (closed triangles, P = 0.4), 1.5 × 10⁷ re-stimulated T cells (open triangle, P < 0.01), or 4 × 10⁷ re-stimulated T cells (closed square, P < 0.01). Number of mice showing complete regression in each treatment group of 5 mice is indicated in parentheses.

Figure 3

CD8⁺ effector T cells can be hyperexpanded through repetitive anti-CD3 stimulation. (A) CD62L^low TDLN cells were restimulated with anti-CD3 mAb for 14 hrs every 7 days starting on day 23 of culture and overall proliferation was measured for three independent experiments. (B) T cells were harvested on day 50 of culture and adoptively transferred to hosts bearing either MCA 205 or MCA 207 3-day pulmonary metastases (P < 0.01 for MCA 205 tumors treated with either 6 × 10⁶ or 2 × 10⁷ compared with all other groups). (C) Mice bearing 3-day s.c tumors were treated with 5 Gy TBI then received adoptive transfer of the indicated number of T cells hyperexpanded for 50 days (P = 0.06 for 4 × 10⁷ cell dose) (D) Mice bearing 3-day i.c. tumors were treated with 5Gy TBI then received adoptive transfer of the indicated number of T cells hyperexpanded for 50 days. Mice were sacrificed when they developed neurologic symptoms indicating progressive tumor (P = 0.9 for treatment groups versus control).

Figure 4

Hyperexpanded CD4⁺ T cells mediate regression of intracranial or subcutaneous tumors. (A) CD62L^low TDLN cells were depleted of CD8⁺ cells prior to anti-CD3 activation and were maintained in medium with IL-2 (4 U/ml) plus IL-7 (10 ng/ml) or alternatively with IL-7 (10 ng/ml) plus IL-23 (2 ng/ml) and were restimulated for 14 hrs with anti-CD3 mAb at the indicated time points. The total proliferation with indicated losses due to AICD or re-purification of CD4⁺ cells is indicated. (B) Mice bearing 3-day s.c.tumors were treated with 5 Gy TBI followed by adoptive transfer of 3 × 10⁷ CD4⁺ T cells culture activated for 43 days and tumor size was measured. On day 37, one mouse from IL-2 + IL-7 group was euthanized due to progressive tumor growth, however complete regression was observed in the remaining 4 mice (P = 0.015 versus control). Complete regression was observed in all five recipients of IL-7 + IL-23 cultured CD4⁺ T cells (P = 0.005 versus control). (C) Mice bearing 3-day intracranial tumors were treated with 5Gy TBI followed by adoptive transfer of 3 × 10⁷ CD4⁺ T cells culture activated for 43 days. Mice were followed for survival (P < 0.01 for both treatment groups versus control). (D) Mice bearing 3-day subcutaneous tumors were treated with 5 Gy TBI followed by adoptive transfer of 4 × 10⁷ CD8⁺ T cells hyperexpanded to greater than 10⁸-fold for 50 days, or 1.5 × 10⁷ CD4⁺ T cells hyperexpanded to greater than 10⁸-fold for 85 days. On day 28, 4 mice from CD8 treatment group (* P = 0.39 versus control) and 2 mice from CD4 treatment group (# P = 0.019 versus control) were euthanized due to progressive tumor growth but complete tumor regression was observed in the remaining mice.

Figure 5

Hyperexpanded CD4⁺ and CD8⁺ T cells produce IFN-γ in response to tumor stimulation. CD62L^low TDLN cells were culture activated with anti-CD3 and IL-2 plus IL-7 for 23 days then were restimulated with anti-CD3 every 7 days. T cells were removed from culture on day 8, on day 36 for CD8⁺ cultures, or day 43 for CD4⁺ cultures. T cells were incubated without additional stimulus to determine spontaneous production of IFN-γ or with single cell digest of MCA205 or MCA207 tumors or with immobilized anti-CD3 mAb and Brefeldin A was added at 5 hrs and cells were harvested after 14 hrs. Intracellular IFN-γ was determined by FACS and the percentage of T cells is indicated.