Perspective on Potential Clinical Applications of Recombinant Human Interleukin-7

Abstract

Interleukin-7 has critical and nonredundant roles in T cell development, hematopoiesis, and postdevelopmental immune functions as a prototypic homeostatic cytokine. Based on a large body of preclinical evidence, it may have multiple therapeutic applications in immunodeficiency states, either physiologic (immuno-senescence), pathologic (HIV) or iatrogenic (postchemotherapy and posthematopoietic stem cell transplant) and may have roles in immune reconstitution or enhancement of immunotherapy. Early clinical development trials in humans show that, within a short time, rhIL-7 administration results in a marked preferential expansion of both naive and memory CD4 and CD8 T cell pools with a tendency toward enhanced CD8 expansion. As a result, lymphopenic or normal older hosts develop an expanded circulating T cell pool with a profile that resembles that seen earlier in life with increased T cell repertoire diversity. These results, along with a favorable toxicity profile, open a wide perspective of potential future clinical applications.

Background

IL-7 is a multifunctional, homeostatic cytokine first isolated as a 25 KDa glycoprotein produced by a murine bone marrow stromal cell line.¹ IL-7 is not produced by lymphocytes but, rather, by bone marrow stroma² as well as other cell types including thymic stroma, keratinocytes, neurons, antigen presenting cells, lymph node follicular dendritic cells, and endothelial cells. IL-7 signals through a heterodimer involving IL-7Rα and the common γ chain (γc). IL-7Rα is shared with TSLP and the γc receptor is shared with IL-2, IL-4, IL-9, IL-15, and IL-21. IL-7 plays a critical, nonredundant role in the development of T cells. This is directly demonstrated in murine models wherein IL-7R knockout mice have arrest of T cell development at a double positive stage³ and IL-7 deficient mice are profoundly lymphopenic with thymic cellularity reduced 20-fold.⁴ In contrast, mice transgenic for IL-7 develop T cell lymphoproliferative/autoimmune diseases and T cell lymphomas.^5,6 The effects of IL-7 on T cell development are multifactorial, involving antiapoptotic and proliferative effects on developing lymphocytes,^7,8 promotion of V(D)J rearrangement of T cell receptor genes,⁹ and provision of trophic and costimulatory signals for mature T cells.¹⁰

In human T cell development, IL-7’s critical role is confirmed by analysis of three groups of patients with severe combined immunodeficiency (SCID) who have mutations involving the IL-7 receptor or its signaling pathway. Children with X-linked SCID (T-NK cell deficient, but spared B cells) have a defect of the γ-chain of the IL-7 receptor,¹¹ children with autosomal recessive SCID (T-NK cell deficient but spared B cells) not infrequently show mutations of the Jak-3 tyrosine kinase,^12,13 and kindreds with a defective α-chain of the IL-7 receptor are severely T cell deficient, but have normal NK cells and B cells.¹⁴ Current concepts hold that the T cell deficiency in each of these clinical entities relates to absent or defective IL-7 signaling during T cell development, whereas NK cell deficiency results from absent or defective IL-15 signaling, which coexists when the genetic defect involves the γ-chain of the IL-7 receptor or the Jak-3 tyrosine kinase.

With regard to B cell development, IL-7 was first recognized as an important maturation and differentiation factor for pre-B cells in murine models¹⁵ and IL-7 is an essential factor for supporting B lymphopoiesis ex vivo.² Moreover, IL-7 transgenic mice show expansion of immature B cells,^5,6 and humans treated with rhIL-7 show expansions in immature B cells within the bone marrow.¹⁶ However, IL-7 does not appear to be essential for human B cell development, since patients with SCID due to γc,JAK3, or IL-7Rα mutations, can have normal or even elevated numbers of peripheral blood B cells.^11–14 Thus, while IL-7 participates in normal B cell development ex vivo,¹⁷ it does not appear to be strictly required for B cell development in humans. IL-7 also stimulates egress of primitive hematopoietic cells from bone marrow, and it has been used successfully as a mobilization agent in mice, resulting in long lasting, full tri-lineage engraftment in mice transplanted with rhIL-7 mobilized peripheral blood, a property that may be clinically exploitable.¹⁸

In addition to its critical role in T cell lymphopoiesis and its effect on developing B cells, IL-7 also plays a central role in peripheral T cell homeostasis. As discussed above, IL-7R shares the receptor common γ-chain (CD132) with several other cytokines. Within this family, cytokines can be classified as activating versus homeostatic. IL-2 is a prototypic activating cytokine, while IL-7 is a prototypic homeostatic cytokine. IL-2 selectively signals activated T cells, is secreted by activated T cells and IL-2 signaling upregulates its own receptor (IL-2Rα; CD25) thus amplifying the IL-2 response during immune activation. In contrast, IL-7Rα (CD127) is expressed on resting T cells but is downregulated following signaling by IL-7 itself, other prosurvival cytokines (IL-2, IL-4, IL-6, IL-15)¹⁹ or following T cell receptor (TCR) ligation. This tight regulation of IL-7Rα expression is congruent with the homeostatic role of IL-7, as it presumably prevents T cells that have already received a prosurvival signal from competing with other cells for its utilization.²⁰ Moreover, while production of activating cytokines occurs in the context of immune activation, IL-7 is continuously produced and available to resting T cells within the lymphoid niche. This continuous availability of IL-7 provides essential trophic signals for homeostatic proliferation and survival of naive T cells, since naive T cells adoptively transferred into an IL-7 deficient host rapidly disappear.²¹

Many studies have documented that IL-7 therapy can dramatically increase peripheral T cell numbers, primarily through augmentation of homeostatic peripheral expansion.^19,22,23 Briefly, homeostatic peripheral expansion is T cell receptor driven cycling, mediated primarily by low-affinity antigens. While augmentation of thymic output has also been recently suggested,²⁴ it remains controversial and very difficult to establish in the context of human trials. Regulatory CD4⁺CD25^hi T cells (Tregs) express low IL-7Rα levels^25,26 and unlike IL-2, IL-7 therapy expands total CD4⁺ T cells without expanding Tregs.^16,27 IL-7 does not play a major role in Treg development, maintenance, and expansion; and in fact, recent evidence suggests that IL-7 may be capable of down modulating Treg activity.²⁸ In summary, in addition to potent effects on developing lymphocytes, IL-7 is required for maintenance of mature T cell populations and supraphysiologic levels of IL-7 expand peripheral T cell populations through a process termed homeostatic peripheral expansion.

Potential Clinical Applications of IL-7 as an Immunorestorative

As individuals age, the adaptive immune system relies increasingly on the recruitment of memory cells to elicit immune responses. This is because the pool of naive T cells (i.e., the source of the wide diversity of specificities for antigen) decreases considerably with age. Furthermore, immune injury, whether physiologic (thymic involution with advancing age, immuno-senescence), pathologic (e.g. progressive immune depletion with HIV infection), or iatrogenic (following immune depleting therapy such as chemotherapy or irradiation), induces profound limitations in the natural pathways of T cell immune reconstitution. Immune reconstitution occurs through two primary pathways. The thymic pathway, which predominates in children, generates new T cells from pluripotent hematopoietic stem cells that home to the thymus and undergo expansion, differentiation, and selection. The resulting T cells, which bear a naive phenotype, display a diverse T cell receptor (TCR) repertoire and are poised to recognize an array of foreign antigens. In contrast, thymic-independent homeostatic peripheral expansion predominates in adults.²⁹ Homeostatic peripheral expansion results in a skewed T cell repertoire, which is poorly diversified and limited mostly to T cells that encounter their specific antigen during the period of immune reconstitution. Furthermore, homeostatic peripheral expansion is unable to restore numbers of CD4⁺ T cells to pretreatment levels.³⁰

Deficits in immune reconstitution are evident in multiple clinical settings wherein patients experience lymphocyte depletion. For instance, patients with human immunodeficiency virus infection, patients following allogeneic stem cell transplantation and older patients following high-dose cytotoxic therapy for cancer,^31–37 show incomplete or prolonged periods of lymphopenia before full immune reconstitution. Individuals older than 45 to 50 years of age, who experience lymphocyte depletion, are likely to continue to have profound deficits in naїve T cells for the rest of their lives. While moderate immune competence can be accomplished through thymic-independent homeostatic peripheral expansion, new pathogens, or pathogens with high rates of mutations such as the influenza virus, may be a cause of substantial morbidity. Indeed, immune responses to immunizations such as influenza are diminished in elderly patients and subjects immunized after cancer chemotherapy appear to remain at increased risk of infection compared to controls (relative risk of developing protective titers ranges from 0.55 to 0.75, compared to a risk of 1 for normal control individuals).³⁸ Likewise, in HIV-infected individuals, antibody responses following immunization to T-dependent antigens are significantly decreased and correlate with the CD4 count.^39,40 Finally, cancer patients with limited immune reconstitution may also be at increased risk for tumor recurrence and are poor candidates for active immunotherapy strategies, which could potentially contribute to diminish disease recurrence.³⁵

Because of the prevalence of long-term immune dysfunction in patients with age associated, iatrogenic or virus induced lymphopenia, there is great interest in administering immunorestoratives to hasten the capacity to restore normal immune function. RhIL-7 appears capable of substantially augmenting homeostatic peripheral expansion with preferential expansion of naive T cells, which bear the most diverse T cell receptor repertoires. Indeed, even athymic mice show fully restored immunocompetence with rhIL-7 therapy.⁴¹ Thus, while it is not clear that rhIL-7 can reverse age, disease, or therapy-associated thymic involution, rhIL-7’s capacity to augment naive cell proliferation may accomplish significant diversification of the T-cell receptor repertoire and restore near normal T cell diversity even in the absence of robust thymopoiesis.

Aging, in and of itself, even in the absence of lymphodepleting chemotherapy also poses a risk for diminished immune competence and preclinical data suggests that rhIL-7 therapy could be therapeutic in this setting. Age-associated abnormalities of both T cell and B cell compartments have been well described,^42,43 and humoral immune responses and immunoglobulin class switch are downregulated in aged mice and humans.^44,45 There are many examples of a significant decrease in vaccine responses in the elderly population (tetanus and tick-borne encephalitis,⁴⁶ pneumococcal vaccine,⁴⁷ influenza).^48–53 Therefore, methods for improving vaccine efficiency in aged populations are critically needed. Given IL-7’s potent capacity to augment responses to immunization and to augment repertoire diversity, it is plausible to consider IL-7 therapy as a means for augmenting vaccine responsiveness in elderly populations.

Potential Role for IL-7 in Tumor-directe Immunotherapy

By enhancing immune reconstitution or expanding the immune cell repertoire, IL-7 could also have a significant role in enhancing immunotherapy for cancer.^19,22,23 IL-7 augments effector and memory responses to vaccination in mice⁵⁴ with preferential enhancement of responses to weak subdominant antigens, and improves survival of the CD8⁺ memory cell pool. In preclinical models, IL-7 therapy augments antitumor responses leading to improved survival when combined with antitumor vaccines.^54,55 When combined with tumor cell immunotherapy, IL-7 significantly prolongs the survival of tumor-bearing mice. This enhanced antitumor protection correlates with an increased number of activated dendritic cells and T cells in lymphoid tissues and increased activated effector T cells in the tumor microenvironment.⁵⁵

The role of IL-7 in immunotherapy may go beyond the well-described enhancement of T cell numbers and T cell repertoire diversity, decreased apoptosis and increased sensitivity to CD3 trigger.¹⁶ In a murine tumor model,²⁸ IL-7 enhanced cytotoxic activity, increased the number of IL-17 producing CD4⁺ T cells, and increased serum cytokine levels (IL-6, IL-1α, IL-1β, IL-12, tumor necrosis factor-α, C-C chemokine ligand-5 (RANTES), macrophage inflammatory protein-1α). Moreover, IL-7 appeared to inhibit Treg function, and increased refractoriness to inhibitory signals by abrogating TGF-β induced inhibition of CD8⁺ T cell proliferation and mediating anti-TGF-β effects through down modulation of Cbl-b expression. Finally, previous work has demonstrated that IL7-Rα⁺ expression on activated T cells is associated with increased central memory cell generation⁵⁶ and IL7-Rα⁺ expression on adoptively transferred antigen-specific T cells correlates with better survival in vivo.^57,58 Thus, it is possible that rhIL-7 therapy could augment the effectiveness of adoptive T cell therapy for cancer by improving survival of IL-7-Rα⁺ central memory populations and diminishing the competitiveness of senescent IL-7-Rα⁻ populations. Finally, recent studies have suggested that rhIL-7 can diminish PD-1 expression on activated CD8⁺ populations,²⁸ an effect which would be predicted to enhance CD8⁺ T cell survival in vivo.

RhIL-7 in Clinical Trials

The first five studies of rhIL-7 initiated in humans evaluated an E. Coli produced, non-glycosylated rhIL-7 (“CYT99 007,” Cytheris Inc., Rockville, MD) using various phase I designs with doses ranging from 3 to 60 μg/kg in three different populations and dose schedules (see Table 1). Two trials involved oncology subjects, two trials involved HIV⁺ subjects, and one trial involved subjects following allogeneic transplantation for nonlymphoid malignancy. In Oncology trial 1, careful immunologic studies were performed on all IL-7 recipients allowing detailed analysis of the effects of rhIL-7 therapy in humans. All subjects treated between 10–60 mcg/kg/dose showed dose dependent increases in circulating absolute lymphocyte counts, and lymphoid organ enlargement (spleen and normal lymph nodes, but not thymus) was seen at the doses of 30 and 60 μg/kg/dose, maximum on day 14, then returning to baseline over several weeks (Fig. 1). In most subjects, CD3⁺ αβ and γδ cells, CD4⁺ and CD8⁺ T cells increased equally, in a clear dose-dependent fashion. The absolute cell numbers peaked one week after the end of treatment (day 21) but remained elevated for up to 2 months following the end of treatment. Similarly, increased lymphocyte counts persisted for 24 and 48 weeks after the last administered rhIL-7 dose in HIV individuals with the longest follow-up.⁵⁹ There was no correlation between subject age and magnitude of the increase in CD3⁺, CD4⁺, or CD8⁺ T cells.

TABLE 1.

Summary of Clinical Studies with rhIL-7 “CYT 99 007”

TRIAL	“Oncology 1”	“Oncology 2”	“HIV 1”	“HIV 2”	“Allogeneic transplantation”
Institution	NCI; ETIB, POB	NCI; SB	Case Western; Cleveland, OH, NIAID; Bethesda, MD	Hôpital Henri-Mondor, Creteil, France	MSKCC
	Bethesda, MD	Bethesda, MD			New York, NY
Investigator	Sportès, C.	Rosenberg, S.	Lederman, M., Sereti, I.	Lévy, Y	van den Brink, M.
Subjects	Refractory malignancy CD3 > 300/mm3	Refractory metastatic melanoma	HIV: HAART > 12 mo. CD4: > 100/mm3	HIV : HAART > 12 mo. CD4: 100–400/mm3	Myeloid leukemia post allogeneic transplantation
Number of subjects	16	12	17 (stratum1) 8 (stratum 2)	14	1
Design	Phase I: dose escalation	Phase I: dose escalation (coadministration of gp 100/MART peptide vaccines)	IL-7/placebo (random 3 to 1) 2 strata: HIV RNA: • < 50 or • 50–50,000 copies	Phase I: dose escalation 2 strata: CD4/mm3 • 100–200 • 200–400	Phase I: dose escalation
Doses/schedule	• 3, 10, 30, 60 μg/kg/dose • every other day; • 8 doses (2 weeks)	• 3, 10, 30, 60 μg/kg/dose • 3 times a week • 8 doses (3 weeks)	• 3, 10, 30, 60 μg/kg/dose • single dose	• 3, 10 μg/kg/dose • 3 times a week • 8 doses (3 weeks)	• 3 μg/kg/dose • 3 times a week • 8 doses (3 weeks)
Reference	[16]	[27]	[60]	[59]

NCI: National Cancer Institute; ETIB: Experimental Transplantation and Immunology Branch; POB: Pediatric Oncology Branch; SB: Surgery Branch; NIAID: National Institute of Allergy and Infectious Diseases; MSKCC: Memorial Sloan-Kettering Cancer Center.

Figure 1.

Effects of rhIL-7 Therapy on Circulating lymphocytes and Spleen Size. RhIL-7 injections indicated by tick marks on X-axis along with baseline, days 7, 14, 21, and 28 data points (additional data points are shown for total lymphocytes count only: day 1 for all and day 55–90 for subjects treated with 30 or 60 μg/kg/d). Mean value for each cohort (± SEM) are plotted: 3 μg/kg/d (black); 10 μg/kg/d (green); 30 μg/kg/d (red); 60 μg/kg/d (blue). (A) Absolute lymphocyte count from complete blood counts (left panels) and percent change in absolute numbers over baseline (right panels) for CD3⁺/CD4⁺ and CD3⁺/CD8⁺ cells the respective subsets are shown. (B) Spleen size: percent changes from the pretherapy bidimensional product by CT scan. (C, D, and E) CD3⁺/CD4⁺ (left panels) and CD3⁺/CD8⁺ subsets (right panels). (C) “Ki-67”: percentage of cells expressing Ki-67 (flow cytometry). (D) “IL-7Rα” expression as mean fluorescence intensity via flow cytometry. (E) “bcl-2” expression as mean fluorescence intensity (after subtracting background staining for each subset) via flow cytometry.

The effects of rhIL-7 on T cell expansion could be attributed to a combination of increased cell cycling and diminished programmed cell death but were self-limited by downregulation of IL-7Rα. At baseline, 1%–3% of circulating CD4⁺ and CD8⁺ T cells were in cycle (expressing Ki-67). Therapy resulted in a dramatic, dose-dependent increase in cycling frequency: in subjects treated with 60 μg/kg/dose, >40% of CD4⁺ peripheral T cells and >55% of CD8⁺ peripheral T cells expressed Ki-67 at day 7. T cell cycling substantially declined by day 14, coincident with maximum IL-7Rα downregulation despite sustained pharmacologic serum IL-7 levels (continued rhIL-7 administration through day 14). As a surrogate for decreased apoptosis, bcl-2 expression was increased and remained elevated throughout the rhIL-7 administration, likely contributing to the increase in T cell numbers. Indeed, at the time of peak lymphocyte expansion, one week after the end of the treatment (day 21), cell cycling had returned to baseline values.¹⁶

Detailed studies of T cell subsets¹⁶ showed that rhIL-7 preferentially expanded the CD4⁺ recent thymic emigrants (CD4⁺/CD31⁺/CD45RA⁺), naive (CD4⁺/CD27⁺/CD45RA⁺), and central memory (CD4⁺/CD45RA⁻/CCR7⁺) T cells as well as CD8⁺ naive (CD8⁺/CD27⁺/CD45RA⁺) T cells (Fig. 2). This T cell expansion resulted in a statistically significant increase in T cell repertoire diversity for both CD4⁺ and CD8⁺ T cells along with a decrease in the proportion of terminally differentiated effector T cells. It is also noteworthy that rhIL-7 preferentially expanded naive and central memory subsets, both of which are important in initiating lymph node germinal center formation. Tregs showed markedly less proliferation and expansion compared to other CD4⁺ T cells, resulting in a decrease in the proportion of circulating regulatory T cells. Therefore, within the short time frame of the study, rhIL-7 therapy induced marked expansion of circulating T cells exhibiting a rejuvenated profile resembling that seen early in life with minimal Treg expansion. These lymphocytes remained functional with conserved or increased in vitro responsiveness to anti-CD3 stimulation. Ongoing studies are underway to determine whether combining antiviral therapy with rhIL-7 may augment viral clearance in hepatitis C infection and whether rhIL-7 therapy can safely augment immune reconstitution following T cell depleted allogeneic SCT.

Figure 2.

RhIL-7 induces preferential expansion of Naive T cells and CD4⁺ Memory T Cells (with cycling and bcl-2 expression increase across subsets).¹⁶ Subjects treated at 3 μg/kg/d (white), 10 μg/kg/d (speckled), 30 μg/kg/d (grey) and 60 μg/kg/d (black). The time points shown represent the point of maximal increase for each parameter: (A) Percent increase in absolute circulating cell number (/mm³) over pretherapy value for each subset at Day 21 following rhIL-7. (B) Net increase from baseline in the proportion of Ki67⁺ cells in each subset at day 7 of rhIL-7 therapy. (C) Net increase in bcl-2 mean fluorescence intensity in each subset at day 14. (D) Net increase in cell number at day 21 in subsets defined by CCR7 and CD45RA surface expression as follows: Naive (CCR7⁺CD45RA⁺), central memory (CCR7⁺CD45RA⁻), effector memory (CCR7⁻CD45RA⁻), and effector RA (CCR7⁻CD45RA⁺).

Summary

RhIL-7 has now entered clinical trials and shows clear evidence for biologic activity that largely mirrors the results founds in mice and primates. IL-7 potently expands T cell populations, predominantly through enhanced peripheral T cell cycling. As a result, lymphopenic hosts show normalization of T cell numbers with rhIL-7 therapy, and normal hosts show supranormal T cell numbers with rhIL-7. In general, both CD4 and CD8 populations are expanded in vivo, with a tendency toward enhanced CD8 expansion. Both naive and memory subsets undergo expansion, but there appears to be a preferential expansion of naive subsets and little if any expansion of senescent memory populations. T cell receptor repertoire diversity can be enhanced by rhIL-7 therapy, even in the absence of direct thymopoietic effects. Tregs are not targeted by rhIL-7, leading to a relative reduction in Treg frequency when rhIL-7 therapy is administered. RhIL-7 shows promise as an immunorestorative for aged individuals or individuals who have experienced iatrogenic or disease induced lymphocyte depletion. It also is active as a vaccine adjuvant in preclinical models, and clinical trials are underway to determine whether it may augment viral clearance in the context of chronic viral infection when used in conjunction with antiviral therapy. Future studies will seek to determine whether rhIL-7 may enhance the effectiveness of tumor vaccines and/or adoptive immunotherapy for cancer.