Adoptive transfer Tc17 effectors treatment leads to regression of melanoma tumors
We assessed the therapeutic efficacy of in vitro generated OVA-specific Tc17 effector T cells to control the growth of small (110 mm3) and large (532 mm3) well-established OVA-expressing melanoma tumors. Mice with small tumors received varying numbers of Tc17 cells at day 7 after tumor implantation. One hundred percent of the mice treated with 1 × 106 (open inverted triangles p = 0.0006, two tailed), 5 × 106 (closed diamonds p = 0.0465 two tailed), and 107 (open circles p = 0.0426, two tailed) Tc17 cells completely suppressed tumor growth (), whereas 5 × 105 effectors (closed triangles p = 0.0003, two tailed) were still suppressive and 105 (closed squares p = 0.145 two tailed) had no effect. As expected, control groups treated with LCMV-specific 5 × 106 Tc17 cells from P14 mice or PBS () also developed large tumor masses.
FIGURE 1 A, Tc17 cells control the growth of established melanoma tumor. Seven groups of C57BL/6 mice were injected with 2 × 105 OVA-expressing B16 cells at day −7. At day 0 five groups of mice were treated with different numbers of Tc17 generated (more ...)
We also tested whether transferred Tc17 effector cells have the capacity to control larger melanoma tumors. 5 × 106 Tc17 cells (closed triangles) were adoptively transferred into tumor-bearing mice at day 12, rather than day 7. The injected mice still showed an impressive tumor reduction at day 4 after T cell transfer () when compared with mice treated with LCMV-specific Tc17 cells (open circles) or with PBS (p = 0.018, two tailed).
The 5 × 106 Tc17 cells were also able to prevent tumor growth in a prophylactic setting. Tc17 cells were injected into recipient mice at day −1 and challenged with 2 × 105 B16-OVA tumor cells at day 0. One hundred percent of mice that received Tc17 cells were tumor-free at day 160, whereas some of the mice from the control group had a palpable tumor at day 5 and all had tumors by day 6 () and were sacrificed at day 15 for humane reasons. Mice preinjected with Tc17 effectors that had prevented tumor growth were subsequently rechallenged twice with 2 × 105 B16-OVA tumor cells and there was no evidence of tumor growth during 5 mo. Thus, the prophylactic transfer of OVA-specific Tc17 effector cells before tumor challenge induced a strong protective and long-lasting memory response that efficiently killed melanoma cells.
In other studies, we could find no evidence of epitope spreading in that the parent tumor grew at the same rate on mice actively rejecting B16-OVA as on control mice (data not shown).
Control of tumor growth is partially dependent on IL-17
Tc17 cells are so named because they produce IL-17A and F, but they also secrete many other potent cytokines, including TNF, IL-21, and IL-22. To evaluate the role of IL-17 derived from Tc17 cells, OVA-specific, Tc17 cells were transferred into tumor-bearing, IL-17RA–deficient mice and the control of tumor growth was compared with that in wild-type hosts that had normal expression of IL-17RA on hematopoietic and stromal cells as shown in . To make a quantitative comparison, we compared the percent reduction in tumor volume at day 6. The capacity to control tumor growth by transferred Tc17 cells in mice lacking IL-17RA was modestly impaired, inducing a 54–66% reduction in tumor growth, contrasting with a more extensive reduction in the tumor mass by Tc17 cells transferred to B6 mice (82–84% reduction) (p = 0.016, two tailed). The control of tumor growth by Tc1 effectors was not significantly affected by the absence of IL-17RA (p = 0.717, two tailed) and a similar protection was observed in wild-type and IL-17RA–deficient recipients after Tc1 transfer. The tumors grew at different rates in the wild-type and IL-17RA mice and we therefore compared the tumor growth in the Tc17-treated wild-type mice with that in a wild-type control and the tumor growth in the Tc17-treated IL-17RA−/− mouse with the IL-17RA control. IL-17RA is the receptor for both IL-17A and F and the ability of the Tc17 cells to still induce a significant reduction in the control of tumor growth, in the absence of IL-17RA, suggests that other cytokines are crucial for reducing tumor growth and likely candidates include TNF and IFN-γ, as addressed in experiments below.
Tc17 effectors are less able to control tumor growth in IL-17RA–deficient host but show considerable residual antitumor activity
Control of tumor growth by Tc17 effectors is independent of perforin expression but is diminished in IFN-γ– and TNF-deficient Tc17 effectors
CTL activity in Tc1 cells is shown by with the capacity to kill target cells in vitro through perforin- and granzyme-dependent mechanisms but we showed previously that this activity is not important for control of tumor growth in vivo (14
), whereas IFN-γ was essential. The Tc17 effector cells lack the usual CD8 effector mechanisms but they produce several cytokines in addition to IL-17, including IL-21 and IL-22 (not examined in this study), and proinflammatory cytokines that help to control tumor growth by activating IFN-γ– and TNF-responsive populations, including macrophages, neutrophils, and T cells.
To test the participation of some of these different CD8 effector molecules in Tc17-mediated effects, we generated Tc17 cells using mice deficient in TNF, IFN-γ, or perforin and tested whether they could still control tumor growth. It was clear that Tc17 cells do not need perforin to reduce tumor growth (, p
= 0.08, two tailed); however, IFN-γ (, p
= 0.006, two tailed; , p
= 0.019, two tailed) and TNF (, p
= 0.028, two tailed; , p
= 0.05, two tailed) production by Tc17 CD8 T cells are partially required for inducing a complete tumor rejection. In , the tumor growth was plotted against time for a single dose (5 × 106
) of transferred cells, but in , we determined the percent reduction in tumor volume at day 6 and plotted it against the log of the number of adoptively transferred wild-type and cytokine-deficient cells as described previously (14
). To test whether cytokine produced by host cells is also important for controlling tumor growth, we transferred different numbers of wild-type or cytokine-deficient Tc17 cells into wild-type C57BL/6 and into TNF- or IFN-γ–deficient C57BL/6 mice and calculated the percentage of reduction of tumor growth at day 6 after T cell transfer. It was clear that the IFN-γ derived from host cells is not important because IFN-γ produced by Tc17 cells was sufficient to control tumor growth, even in the absence of a host source (). Tumor reduction was dependent on donor TNF and there appeared to be some dependence on host TNF, to efficiently control tumor growth (). The need for TNF and IFN-γ production by donor Tc17 cells was demonstrated by the fact that ~10-fold more cytokine-deficient donor cells were needed to achieve the same level of tumor control as seen with wild-type donor cells ().
FIGURE 2 A, IFN-γ and TNF, but not perforin, are required for complete control of tumor growth. Groups of tumor-bearing mice were treated with 5 × 106 Tc17 cells generated from OT-1 (closed squares), perforin- (closed triangles), IFN-γ– (more ...)
The finding that IFN-γ was required, at first site, seemed surprising because previous studies demonstrated that Tc17 cells lack the ability to secrete IFN-γ after restimulation when generated in vitro. However, they regain the ability to produce IFN-γ some days after injection back into the animal (24
) and most IL-17 secreting cells become double producers. IFN-γ is a cytokine fundamental for induction of IFN-γ induced chemokines like CXCL9, CXCL10, and CXCL11, which we hypothesized could be important in the recruitment of effector T cells to the tumor and help to eliminate malignant cells and a similar role can be envisioned for TNF.
Tc17 cell transfer induces recruitment of donor and host lymphocytes to the tumor by IFN-γ– and TNF- dependent mechanisms
Tc17 cells express a number of proinflammatory cytokines that can, by analogy with Th17, be expected to induce the formation of chemotactic factors involved in the recruitment of lymphocytes to the tumor and we had shown previously that both TNF and IFN-γ played some role in the control of tumor growth. We next analyzed the kinetics of cell migration into the tumor in mice receiving wild-type or cytokine-deficient Tc17 cells.
The 5 × 106 Tc17 cells derived from CD45.2+ OT-1 wild-type, or TNF- or IFN-γ–deficient OT-1 mice were injected into CD45.1. C57BL/6 mice 7 d after the injection of tumor, as before. Cohorts of mice were sacrificed at the times indicated, the tumors were removed and the number of infiltrating donor and host cells were determined by flow cytometry, as described in Materials and Methods. Mice that received wild-type Tc17 cells showed a peak in the number of tumor infiltrating tetramer+ donor T cells at day 4 after transfer (). A somewhat smaller peak was seen in IL-17 secreting donor cells (), donor IFN-γ–secreting cells () and donor TNF-secreting cells () at day 4. It was striking that mice injected with IFN-γ (closed squares) or TNF (closed inverted triangles) deficient donor cells showed much smaller numbers of donor infiltrating cells of all types and a delayed kinetics in each of these categories ().
FIGURE 3 Tc17 therapy induces an increased recruitment of immune cells into tumor. Tumor-bearing mice were treated with 5 × 106 Tc17 wild-type (closed triangles), IFN-γ– (closed squares), and TNF- (inverted closed triangles) deficient OT-1 (more ...)
This same pattern was also seen for the recruitment of CD45.1 host cells into the tumor as shown in , panels e–g. Large numbers of host tetramer positive cells were seen at day 6 (, panel e), whereas smaller numbers of host IL-17–secreting cells peaked at day 4 (, panel f). There was an exception for the endogenous CD8+ CD45.1+ TNF-deficient cells, which had a totally different trend. They peaked at day 2 after transfer of CD45.2 Tc17 cells, began to decline at day 4 posttransfer and they were almost absent at day 6 posttransfer (, panel h)
It was clear that transfer of Tc17 cells, unable to produce TNF or IFN-γ, led to a delayed and less efficient recruitment of both donor and host cells, which correlated with the less effective control of tumor growth. This suggests that both cytokines are important for early recruitment of effector CD8 T cells.
Transfer of effector Tc17 cells induces recruitment of neutrophils and macrophages to the tumor by IFN-γ–and TNF-dependent mechanisms, but does not alter the recruitment of dendritic cells, eosinophils, NK cells, NKT cells, and B cells
We used the same protocol as above, injecting Tc17 effectors prepared from wild type, IFN-γ and TNF deficient mice into tumor-bearing mice to determine the numbers of activated dendritic cells, NK, NKT, eosinophils, macrophages, neutrophils, and B cells recruited to the tumor site. We found no statistically significant difference in the total numbers of any of the leukocyte populations between the three groups for NK, NKT, eosinophils, and B cells (data not shown). But transfer of Tc17 effectors induced a statistically significant increase in the numbers of tumor-infiltrating neutrophils and macrophages in the treated mice. Recruitment of both neutrophils and macrophages to tumors followed the same kinetics as the T cell response, reaching its peak at day 4 in mice that received wild-type Tc17 cells (). Again, it was evident that TNF and IFN-γ derived from the Tc17 donor cells were important for the attraction of these cells to the tumor site. Mice that received Tc17 cells that lack TNF and IFN-γ showed a drastic impairment of neutrophil and macrophage migration to the tumor at the peak of the response. It is noteworthy that the somewhat smaller numbers of Tc1 effectors () required for the same degree of control of tumor growth did not induce significant increase in the recruitment of either neutrophils or macrophages. It was curious that the peak in macrophage accumulation does not correlate with peak in the chemokine messages (see below) that one might have been expected to be responsible for the macrophage recruitment and this discrepancy remains unexplained.
FIGURE 4 Tc17 therapy induces recruitment of macrophages and neutrophils into tumor. A, Tumor-bearing mice were treated with 106 Tc1 or 5 × 106 Tc17-, TNF KO and IFN-γ KO OT-1 cells. Control mice were treated with PBS. Mice were sacrificed at day (more ...)
The relevance of TNF and IFN-γ derived from Tc17 cells in the induction of mRNA expression for TNF, IFN-γ, and IL-17 in the tumor was confirmed by quantitative PCR (), which shows that IFN-γ and TNF production by Tc17 cells is important for expression of mRNA levels of IL-17 within the tumor, as demonstrated by the very low levels of IL-17 mRNA detected in the tumor of mice that received Tc17 cells lacking IFN-γ and the reduced levels seen in tumors infiltrated by effectors lacking TNF (, panel a). In a similar way, absence of TNF or IFN-γ production by Tc17 cells was reflected by low levels of mRNA expression for TNF (, panel b) and IFN-γ (, panel c) at the tumor site suggesting that these cytokines derived from Tc17 cells are also vital for efficient intratumoral mRNA expression of IL-17, TNF, and IFN-γ.
In further analyses, we measured mRNA levels of chemokines by TaqMan PCR, at different times after transfer of Tc17 cells or Tc17 cells deficient in cytokine production and established that mRNA expression for CXCL2 (neutrophil-attracting chemokine, , panel d) and CCL2 (macrophage-attracting chemokine, , panel e) was reduced in tumors of mice treated with Tc17 cells from mice deficient in the production of TNF or IFN-γ as also shown in , panels d and e. These results demonstrate that although IL-17 is the signature cytokine produced by Tc17 cells, the capacity to recruit inflammatory cells to the tumor and induce expression of protective cytokines is linked to their capacity to produce TNF and IFN-γ.
Tc17 cell transfer induces activation of tumor-infiltrating neutrophils, accelerates death of melanoma cells, and is crucial for control of tumor growth
To know more about the biological activity and the activation state of tumor-infiltrating neutrophils, we isolated them from established tumors at day 4 after Tc17 injection and assessed the quality of the preparation by staining cytospin cell suspensions with Abs that recognize neutrophil surface markers, such as Gr-1 and 7/4. Only cells that are double positive for Gr-1 and 7/4 (data not shown) and displayed the characteristic multilobulated nuclei () were seen. To determine the activation state of neutrophils in the tumor, mRNA was isolated from infiltrating neutrophils from each experimental group and the relative changes in mRNA expression were determined by quantitative PCR.
FIGURE 5 Purification and characterization of neutrophils from tumors of mice treated with Tc17. A, Mice with 7 d established tumors were treated with 5 × 106 Tc17 OT-1 cells or PBS and then, tumor infiltrating neutrophils were purified at day 4. Cytospin (more ...)
Transfer of Tc17 cells to tumor-bearing mice was associated with a drastic activation of tumor infiltrating neutrophils, which had an enhanced expression of mRNA for TRAIL, COX2, and iNOS2 as shown in . Thus, Tc17-derived soluble factors are not only important for the attraction of neutrophils to the tumor site, but also for their activation. Message for MPO was not affected.
We also evaluated the killing capacity of neutrophils isolated from Tc17-treated mice by observing neutrophil-tumor cell interactions. GFP+ neutrophils, isolated from treated mice, forming clusters with dying melanoma cells are shown in , upper panel. In contrast only a few neutrophils, isolated from tumors of control group were found in close proximity to live melanoma cells, , lower panel. In the movie, itself, it could be seen that tumor cells died after interaction with the activated neutrophils. To have a more quantitative idea about the killer capacity of activated neutrophils, we collected the remaining supernatants and cells from the Petri dish used for recording neutrophil-melanoma cell interactions and the frequency of dead cells was quantified by flow cytometry. We found 5% of dead tumor cells in cultures that had neutrophils isolated from Tc17-treated mice, compared with 1% of dead tumor cells in cultures containing neutrophils isolated from PBS-treated groups (data not shown). Although the frequency of dying tumor cells was higher in wells that contained neutrophils purified from tumors of Tc17-treated mice, it was not impressive. To confirm that neutrophils were inflicting damage to tumor cells, we recovered melanoma cells from the cultures incubated with neutrophils isolated from Tc17- or PBS-treated groups, injected equal numbers (2 × 105 tumor cells) into B6 mice and monitored the presence or absence of tumors. The PBS group began to develop tumors by day 5 and all had tumors by day 8, whereas in the Tc17-treated group, only one of five mice developed a tumor at day 11 and four of them (75%) remained tumor free until the end of the experiment (day 20 after tumor inoculation) as shown in .
The relevance of neutrophils in the control of tumor growth was confirmed by the elimination of this cell population with the injection of a neutrophil depleting Ab. Treatment with anti-Ly6G resulted in a statistically significant reduction in the capacity of Tc17-treated mice to control tumor growth (, p = 0.0016, two tailed). Similar results were obtained using a different neutrophil depleting Ab (RB6, anti-GR-1), data not shown.
These findings indicate that factors from transferred Tc17 cells are important to recruit and activate neutrophils at the tumor site, making them capable to induce damage to tumor cells and accelerate tumor eradication. Tumor infiltrating-neutrophils are thus a critical component in the induction of Tc17-dependent immunity against melanoma tumors.
The ability of the transferred Tc17 effector cells to attract neutrophils is associated with the production of IL17, TNF, and IFN-γ
In our Tc17 transfer model, we had shown previously that proinflammatory cytokines derived from Tc17 cells induced the production of a variety of chemokines by resident stromal cells, tumor cells, or recently recruited inflammatory cells. It is also likely, however, that Tc17 cells, themselves, could be an additional source of chemokines implicated in the attraction of type I effector cells or neutrophils. To prove Tc17 cells are producing chemokines that attract neutrophils and to evaluate the role of Tc17-associated cytokines in neutrophil chemotaxis, we measured the attraction of neutrophils by cytokine deficient- and wild-type Tc17 stimulated with PMA and ionomycin. As a positive control for neutrophil chemoattraction, we loaded the low chamber with the classic neutrophil-chemoattractant CXCL2. Stimulated Tc17 cells were able to attract neutrophils, but they were less efficient than CXCL2 alone (). In addition, it was clear that TNF and IFN-γ produced by Tc17 are both important for efficient neutrophil attraction, perhaps because they are involved in the autocrine induction of chemotactic factors by Tc17 cells.
FIGURE 6 Attraction of neutrophils by Tc17 cells depends on IL-17, IFN-γ, and TNF production. A, Activated Tc17 effectors attract neutrophils. Mouse bone marrow neutrophils were placed in a transwell chamber containing 2 × 106 in vitro generated (more ...)
It is also likely that cytokines produced by Tc17 cells can activate neutrophils, inducing production of chemotactic factors that can enhance neutrophil chemotaxis. To test this latter possibility, we evaluated the migratory capacity of different cytokine receptor-deficient neutrophils, isolated from bone marrow, toward chemotactic factors produced by stimulated Tc17 cells. Again, neutrophils migrated more efficiently toward CXCL2, compared with the migration promoted by simulated Tc17 cells. We found a reduction in the migratory capacity of neutrophils from cytokine receptor-deficient mice (). This was surprising but suggested that somehow the cytokines produced by Tc17 cells were responsible for improving neutrophil migration, perhaps by induction of chemokine receptors.
It is also possible that the cytokine-activated neutrophils can be a source of chemokines that are important for the attraction of more neutrophils. To test this, we isolated neutrophils from tumors of PBS- and Tc17-treated mice and evaluated the production of different chemokines at the mRNA level. Although CXCL2 was detected in neutrophils activated in vitro (data not shown), neutrophils isolated from Tc17-treated mice produced low mRNA levels for that chemokine. Interestingly, neutrophils isolated from Tc17-treated mice produced chemotactic factors that are important for the attraction of type 1 effector cells. They expressed considerable mRNA levels of CXCL9, moderate mRNA levels of CXCL10 and CCL5 and showed a poor induction of mRNA expression for CCL3 and CCL4 (). These results show that activated Tc17 are able to attract neutrophils and that cytokines produced by Tc17 are somehow stimulating neutrophils to enhance their migratory abilities. In addition, activated neutrophils are a good source of chemokines implicated in the attraction of type 1 effector cells that are crucial for tumor control.
Tc17 cells produce chemokines that attract in vitro generated Th1 cells
Studies have shown that infiltrating CD8 T cells can produce CXCL9, CXCL10, and CCL5 (47
) that can recruit type I effector cells, To demonstrate that in vitro generated Tc17 cells are a source of chemokines, we analyzed expression of CCL3, CCL4, CCL5, CXCL9, and CXCL10 by flow cytometry. We established that all Tc17 cells produced CXCL9 (100%), but only a smaller percentage of Tc17 cells produced CCL4 (87.8%), CCL3 (73.7%), CXCL10 (64.8%), or CCL5 (34.4%) as shown in . To demonstrate that chemokines produced by activated Tc17 cells were functional, we set up a chemotaxis assay for in vitro generated Th1 cells. Stimulated Tc17 cells were able to attract in vitro generated Th1 cells as shown in and it was clear that blocking Abs were able to reduce migration of Th1 cells. Anti-CCL3 (p
= 0.050, two tailed), anti-CCL4 (p
= 0.028, two tailed), and anti-CXCL10 (p
= 0.047, two tailed) showed the greatest reduction in the CI suggesting that these chemokines were the most effective for recruiting Th1 cells. In contrast, Abs directed against CCL5 (p
= 0.678, two tailed) and CXCL9 (p
= 0.196, two tailed) had less impact on Th1 migration. These results show that activated Tc17 cells are able to recruit Th1 cells through the production of chemokines induced by proinflammatory chemokines.
FIGURE 7 Chemokine production by Tc17 effectors. A, Tc17 cells produce chemokines involved in the attraction of Th1 cells. Four-day in vitro generated Tc17 OT-1 cells were stimulated with SIINFEKL peptide for 3 h in RPMI media containing brefeldin A. CD8 T cells (more ...)
Rapid recruitment of CXCR3+ effector cells is key for the early control of melanoma growth
Tc17 cells and neutrophils are a considerable source of IFN-γ–induced chemokines and thus they are possibly attracting CXCR3+ T cells that can enhance tumor immunity. To confirm that CXCR3+ populations are important for the control of experimental melanoma, we transferred Tc17 cells to CXCR3-deficient mice and monitored tumor growth. Even though CXCR3-deficient mice that received Tc17 cells were still able to eliminate the tumor cells (, closed circles, p = 0.035, two tailed), there was some indication of impaired immunity at early stages of tumor growth. This finding suggests that recruitment of CXCR3+ cells may be important. The control of tumor growth at later times seems to be associated to the massive numbers of infiltrating neutrophils detected by FACS at day 11 after Tc17 cell transfer (data not shown). Tc17 cells and neutrophils produce chemokines that can attract CCR5+ cells too, which can be important for generation of protective tumor immunity. To explore this alternative hypothesis, we transferred Tc17 cells to CCR5-deficient mice. In the absence of CCR5 expression, tumor volume was a slightly bigger, but significantly different from tumor volume in wild-type mice (p = 0.0032, two tailed) (). This means, CCR5 is minimally important for tumor immunity in our transfer model and CCR5 absence is compensated by infiltration of tumors by neutrophils and CXCR3 effector T cells (data not shown). These results suggest that chemokines produced by Tc17 and neutrophils, specific for CXCR3 and CCR5 receptors, are required for the recruitment of effector cells that maintain optimal levels of chemokines and cytokines crucial for sustaining waves of migration of new inflammatory cells armed with antitumor cytokines.
FIGURE 8 Early recruitment of CXCR3+ effector cells is important for rapid induction of tumor immunity. A, At day −10 groups of C57BL/6-(triangles) or CXCR3-deficient (circles) mice were injected with 2 × 105 OVA-expressing B16 cells. At day 0 (more ...)
Tc17 effectors are less effective than Tc1 effectors in the control of tumor growth
Finally, we sought to evaluate the relative effectiveness of Tc17 and Tc1 effectors in the control of tumor growth. We had previously shown that adoptive transfer of varying numbers of Tc1 or Tc2 effectors into tumor-bearing mice could control the growth of established tumors but that Tc1 were more effective than Tc2 in that only one twentieth of the number of Tc1 cells were required to effect the same level of control. In this paper we sought to make a similar comparison between the effectiveness of Tc17 and Tc1 effectors. Varying numbers of Tc17 and Tc1 effectors were transferred into tumor-bearing mice 7 d after tumor injection and the size of the tumors monitored for the next 20 d (data not shown). Comparisons proved rather hard to assess as the tumor mass grew more rapidly at first in the case where tumor growth was subsequently controlled so it was a problem at which point to make the comparison. Those treatments that did well at day 8 appeared to do poorly at day 3 with the crossover point around day 5. The results were somewhat variable but in an initial experiment 5 × 106 Tc17 effectors were as effective, and 5 × 105 Tc17 cells were less effective, than 5 × 105 Tc1 cells at controlling tumor growth (data not shown), suggesting that Tc17 were roughly comparable in effectiveness with Tc2. A second experiment gave comparable results with 5 × 106 Tc17 being comparable to 5 × 105 Tc1 as shown in .
Our earlier studies had suggested that Tc17 homed well to lung after influenza challenge (24
) and we carried out a second experiment to compare the effectiveness of Tc17 and Tc1 on the control of B16-OVA growing as lung metastases. In this model, it is not possible to follow the growth of the tumor without the sacrifice of the animal, so we determined the relative numbers of effectors required to bring about the same prolongation of survival. It can be seen () that Tc1 were again more effective than cells of the Tc17 subset in bringing about a 20 d prolongation of survival that was achieved with 50-fold fewer Tc1 effectors. Neither of these can be considered to yield a definitive number for the ratio but it is clear that Tc17 or Tc2 effectors administered alone are significantly less effective than Tc1.