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Administration of rhIL-7 in humans increases in vivo TCR repertoire diversity by preferential expansion of naive T cell subsets

Rockefeller University Press
Journal of Experimental Medicine (JEM)
Authors:
Claude Sportes at Augusta University
Claude Sportes
Fran Hakim at U.S. Department of Health and Human Services
Fran Hakim
Sarfraz Memon at National Institutes of Health
Sarfraz Memon
Hua Zhang at SPH Biotherapeutics (HK) Limited
Hua Zhang
  • SPH Biotherapeutics (HK) Limited
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Abstract and Figures

Interleukin-7 (IL-7) is a homeostatic cytokine for resting T cells with increasing serum and tissue levels during T cell depletion. In preclinical studies, IL-7 therapy exerts marked stimulating effects on T cell immune reconstitution in mice and primates. First-in-human clinical studies of recombinant human IL-7 (rhIL-7) provided the opportunity to investigate the effects of IL-7 therapy on lymphocytes in vivo. rhIL-7 induced in vivo T cell cycling, bcl-2 up-regulation, and a sustained increase in peripheral blood CD4(+) and CD8(+) T cells. This T cell expansion caused a significant broadening of circulating T cell receptor (TCR) repertoire diversity independent of the subjects' age as naive T cells, including recent thymic emigrants (RTEs), expanded preferentially, whereas the proportions of regulatory T (T reg) cells and senescent CD8(+) effectors diminished. The resulting composition of the circulating T cell pool more closely resembled that seen earlier in life. This profile, distinctive among cytokines under clinical development, suggests that rhIL-7 therapy could enhance and broaden immune responses, particularly in individuals with limited naive T cells and diminished TCR repertoire diversity, as occurs after physiological (age), pathological (human immunodeficiency virus), or iatrogenic (chemotherapy) lymphocyte depletion.
Effects of rhIL-7 therapy on circulating T cells and spleen. rhIL-7 was administered every other day on day 1–14 (8 injections, indicated by tick marks on X axis), at 4 dose levels: 3 μg/kg/d (dashed line with •; n = 3); 10 μg/kg/d (dotted line with △; n = 3); 30 μg/kg/d (dashed line with □; n = 5); and 60 μg/kg/d (solid line with ○; n = 4). Mean value for each cohort (± the SEM) are plotted at the indicated time points. (A) The absolute lymphocyte count from complete blood counts (left) and flow cytometry–based frequency were used to determine circulating absolute CD3⁺/CD4⁺ and CD3⁺/CD8⁺ counts, absolute numbers ± the SEM (bottom graphs), and percent change in absolute numbers over baseline (top graphs) of the respective subsets shown on day 1 (total lymphocytes only), 7, 14, 21, and 28 for all treated subjects, as well as day 55–90 for subjects treated with 30 or 60 μg/kg/d. Baseline values represent the mean of four separate analyses performed in each subject within 2 wk before initiation of rhIL-7 therapy. (B) Spleen size: bidimensional product by CT scan obtained at the time points shown; percent changes from the pretherapy scan are plotted. (C–F) Baseline (1 value obtained on day 0) and day 7, 14, 21, and 28 data points were generated for CD3⁺/CD4⁺ (left) and CD3⁺/CD8⁺ subsets (right). (C) Ki-67 shows the percentage of cells in the respective subsets expressing Ki-67 via flow cytometry at the time points shown. (D) IL-7Rα expression as MFI via flow cytometry for each subset. (E) IL-7Rα mRNA expression represented as mean percent change from baseline of IL-7-Rα mRNA copies normalized/10⁴ copies of AXTB via Q-PCR in sorted CD4⁺ and CD8⁺ cells. (F) bcl-2 expression as MFI (after subtracting background staining for each subset) via flow cytometry for each subset.
Effects of rhIL-7 therapy on circulating T cells and spleen. rhIL-7 was administered every other day on day 1–14 (8 injections, indicated by tick marks on X axis), at 4 dose levels: 3 μg/kg/d (dashed line with •; n = 3); 10 μg/kg/d (dotted line with △; n = 3); 30 μg/kg/d (dashed line with □; n = 5); and 60 μg/kg/d (solid line with ○; n = 4). Mean value for each cohort (± the SEM) are plotted at the indicated time points. (A) The absolute lymphocyte count from complete blood counts (left) and flow cytometry–based frequency were used to determine circulating absolute CD3⁺/CD4⁺ and CD3⁺/CD8⁺ counts, absolute numbers ± the SEM (bottom graphs), and percent change in absolute numbers over baseline (top graphs) of the respective subsets shown on day 1 (total lymphocytes only), 7, 14, 21, and 28 for all treated subjects, as well as day 55–90 for subjects treated with 30 or 60 μg/kg/d. Baseline values represent the mean of four separate analyses performed in each subject within 2 wk before initiation of rhIL-7 therapy. (B) Spleen size: bidimensional product by CT scan obtained at the time points shown; percent changes from the pretherapy scan are plotted. (C–F) Baseline (1 value obtained on day 0) and day 7, 14, 21, and 28 data points were generated for CD3⁺/CD4⁺ (left) and CD3⁺/CD8⁺ subsets (right). (C) Ki-67 shows the percentage of cells in the respective subsets expressing Ki-67 via flow cytometry at the time points shown. (D) IL-7Rα expression as MFI via flow cytometry for each subset. (E) IL-7Rα mRNA expression represented as mean percent change from baseline of IL-7-Rα mRNA copies normalized/10⁴ copies of AXTB via Q-PCR in sorted CD4⁺ and CD8⁺ cells. (F) bcl-2 expression as MFI (after subtracting background staining for each subset) via flow cytometry for each subset.
… 
PET-CT imaging of the self-limited lymphoid organs' enlargement and increased metabolic activity after rhIL-7 therapy. Shown are representative images on PET-CT scan in one subject (42-yr-old female treated with 60 μg/kg/dose). (left) Imaging on day 14 (last day of rhIL-7 treatment). (right) Day 56 (6 wk after the end of treatment). The full ovals indicate areas of increased size (except for vertebral bodies) and activity of lymphoid organs (left and right axillary adenopathy, spleen, and lumbar spine) at the end of treatment. The dotted contours indicate the thymic area where no increased activity can be demonstrated at day 14. Increased metabolic activity is seen in pink and maximal in yellow areas.
PET-CT imaging of the self-limited lymphoid organs' enlargement and increased metabolic activity after rhIL-7 therapy. Shown are representative images on PET-CT scan in one subject (42-yr-old female treated with 60 μg/kg/dose). (left) Imaging on day 14 (last day of rhIL-7 treatment). (right) Day 56 (6 wk after the end of treatment). The full ovals indicate areas of increased size (except for vertebral bodies) and activity of lymphoid organs (left and right axillary adenopathy, spleen, and lumbar spine) at the end of treatment. The dotted contours indicate the thymic area where no increased activity can be demonstrated at day 14. Increased metabolic activity is seen in pink and maximal in yellow areas.
… 
rhIL-7–expanded T cells show robust proliferative responses to CD3 signaling and are not enriched for FOXP3 expressing cells.(A) PBMCs were collected at the time points shown, cryopreserved, and then thawed before analysis. Proliferation in response to titrated doses of plate-bound anti-CD3 was measured by ³H incorporation, as described in the Materials and methods. Similar increases in CD3-induced proliferation were observed across dose levels, therefore results for all dose levels are pooled (n = 15) and compared with normal donors run simultaneously (solid line with ▪, n = 16). rhIL-7–treated subjects showed increased proliferative responses to anti-CD3 on day 7 (dotted line with ▾), day 14 (dashed line with ♦), and day 21 (dashed line with •) compared with normal donors and on day 7 and 14 compared with baseline values (dashed and dotted line with filled ▴; P < 0.05, two-tailed Mann-Whitney test). (B) FOXP3 mRNA copies per 10⁴AXTB mRNA were quantified from sorted CD3⁺/CD4⁺ cells as detailed in the Materials and methods. No significant change in FOXP3 mRNA was detected between baseline values (white) and values obtained at the time of CD4⁺ expansion (day 14 in gray and day 21 in black; P = NS). Mean ± the SEM of data from subjects enrolled at each dose level are shown: 3 μg/kg (n = 2), 10 μg/kg (n = 3), 30 μg/kg (n = 4), and 60 μg/kg (n = 5). (C) Two-dimensional plots (top) and overlay histograms (bottom) of Ki67 expression in T reg cells and other CD4⁺ and CD8⁺ T cells in an individual subject. Percent Ki-67⁺ cells are indicated in each frame. Pretreatment, shaded histograms; day 7, black; day 14, dark gray; day 21, light gray.
rhIL-7–expanded T cells show robust proliferative responses to CD3 signaling and are not enriched for FOXP3 expressing cells.(A) PBMCs were collected at the time points shown, cryopreserved, and then thawed before analysis. Proliferation in response to titrated doses of plate-bound anti-CD3 was measured by ³H incorporation, as described in the Materials and methods. Similar increases in CD3-induced proliferation were observed across dose levels, therefore results for all dose levels are pooled (n = 15) and compared with normal donors run simultaneously (solid line with ▪, n = 16). rhIL-7–treated subjects showed increased proliferative responses to anti-CD3 on day 7 (dotted line with ▾), day 14 (dashed line with ♦), and day 21 (dashed line with •) compared with normal donors and on day 7 and 14 compared with baseline values (dashed and dotted line with filled ▴; P < 0.05, two-tailed Mann-Whitney test). (B) FOXP3 mRNA copies per 10⁴AXTB mRNA were quantified from sorted CD3⁺/CD4⁺ cells as detailed in the Materials and methods. No significant change in FOXP3 mRNA was detected between baseline values (white) and values obtained at the time of CD4⁺ expansion (day 14 in gray and day 21 in black; P = NS). Mean ± the SEM of data from subjects enrolled at each dose level are shown: 3 μg/kg (n = 2), 10 μg/kg (n = 3), 30 μg/kg (n = 4), and 60 μg/kg (n = 5). (C) Two-dimensional plots (top) and overlay histograms (bottom) of Ki67 expression in T reg cells and other CD4⁺ and CD8⁺ T cells in an individual subject. Percent Ki-67⁺ cells are indicated in each frame. Pretreatment, shaded histograms; day 7, black; day 14, dark gray; day 21, light gray.
… 
rhIL-7 therapy increases TCR repertoire diversity by Vβ family spectratype analysis. (A) Schematic representation of the methodology. Vβ family spectratype analysis was performed for each of 6 subjects (3 of each cohort 3 and 4), separately on CD4⁺ and CD8⁺ cells by RT-PCR on RNA extracted from sorted T cells at baseline and at D21. The distributions of peaks for each Vβ family are graphically represented on a curve. (B) Schematic of divergence score calculation. Divergence from the normal donor standard was calculated for each Vβ family, separately for CD4⁺ and CD8⁺ cells, for each individual, at baseline and at day 21. Two Vβ families (Vβ14 on the left and Vβ23 on the right) are illustrated for the sorted CD8⁺ population in one individual (Pt 11), pre (rows 1 and 3), and post (rows 2 and 4) therapy. Overlays of histograms for the subject (no color) and the normal donor standard (in gray) illustrate how the divergence scores were calculated (see Materials and methods). (C)Divergence scores for CD8⁺ cells in one subject. Divergence scores are shown at baseline (pre) and day 21 (post) for each Vβ family, and a diversity shift was obtained (Pre-Post). The median diversity shift (a positive value indicates a shift toward greater repertoire diversity) and the P value of Wilcoxon pair test comparing the pre and post divergence scores are shown, and then incorporated into the summary in D. (D) Summary of repertoire diversity analysis. for each tested individual (age), shown at baseline (D0) and D21 are as follows: the total naive CD4⁺ and naive CD8⁺ cells counts; the median diversity shift for each CD4 and CD8 subsets of each individual and the P values of the Wilcoxon pair test indicating the likelihood that the calculated change in divergence scores for a particular subject's CD4⁺ or CD8⁺ cells between day 21 and baseline occurred by chance; P values <0.05 indicate a statistically significant increase in diversity toward normality between baseline and day 21 studies.
rhIL-7 therapy increases TCR repertoire diversity by Vβ family spectratype analysis. (A) Schematic representation of the methodology. Vβ family spectratype analysis was performed for each of 6 subjects (3 of each cohort 3 and 4), separately on CD4⁺ and CD8⁺ cells by RT-PCR on RNA extracted from sorted T cells at baseline and at D21. The distributions of peaks for each Vβ family are graphically represented on a curve. (B) Schematic of divergence score calculation. Divergence from the normal donor standard was calculated for each Vβ family, separately for CD4⁺ and CD8⁺ cells, for each individual, at baseline and at day 21. Two Vβ families (Vβ14 on the left and Vβ23 on the right) are illustrated for the sorted CD8⁺ population in one individual (Pt 11), pre (rows 1 and 3), and post (rows 2 and 4) therapy. Overlays of histograms for the subject (no color) and the normal donor standard (in gray) illustrate how the divergence scores were calculated (see Materials and methods). (C)Divergence scores for CD8⁺ cells in one subject. Divergence scores are shown at baseline (pre) and day 21 (post) for each Vβ family, and a diversity shift was obtained (Pre-Post). The median diversity shift (a positive value indicates a shift toward greater repertoire diversity) and the P value of Wilcoxon pair test comparing the pre and post divergence scores are shown, and then incorporated into the summary in D. (D) Summary of repertoire diversity analysis. for each tested individual (age), shown at baseline (D0) and D21 are as follows: the total naive CD4⁺ and naive CD8⁺ cells counts; the median diversity shift for each CD4 and CD8 subsets of each individual and the P values of the Wilcoxon pair test indicating the likelihood that the calculated change in divergence scores for a particular subject's CD4⁺ or CD8⁺ cells between day 21 and baseline occurred by chance; P values <0.05 indicate a statistically significant increase in diversity toward normality between baseline and day 21 studies.
… 
rhIL-7 therapy induces cycling of most T cell subsets, but preferentially expands naive CD4⁺ and CD8⁺ cells and CD4⁺ central memory cells. Representative data from a single subject treated at 60 μg/kg/d are shown. (A) Two-dimensional plots illustrating the rhIL-7 therapy–induced increased frequency of CD4⁺ RTEs (CD31⁺/CD45RA⁺; red boxes in first row of graphs), and changes in CD4⁺ and CD8⁺ naive, memory, and effector subsets (respectively CD45RA⁺/CD27⁺, CD45RA⁻/CD27⁺, and CD45RA⁻/CD27⁻ sections on second and third rows). (B) Overlay histograms of Ki-67 expression on CD4⁺ RTEs (CD31⁺/CD45RA⁺) and CD4⁺ and CD8⁺ naive (CD45RA⁺/CD27⁺), memory (CD45RA⁻/CD27⁺), and effector (CD45RA⁻/CD27⁻) subsets. Percent Ki-67⁺ cells are indicated in each frame. Red lines, pretreatment; blue lines, day 7; green lines, day 14; orange lines, day 21. Note that at the end of treatment (green line), the percentage of Ki67⁺ memory and effector CD8⁺ populations drop to lower levels than the naive CD8⁺. (C) Two-dimensional plots illustrating the rhIL-7 therapy–induced changes in CD4⁺ (top row) and CD8⁺ (bottom row) naive, central memory, effector memory, and effectors (respectively CD45RA⁺/CCR7⁺, CD45RA⁻/CCR7⁺, CD45RA⁻/CCR7⁻, and CD45RA+/−/CCR7⁻ sections).
+2
rhIL-7 therapy induces cycling of most T cell subsets, but preferentially expands naive CD4⁺ and CD8⁺ cells and CD4⁺ central memory cells. Representative data from a single subject treated at 60 μg/kg/d are shown. (A) Two-dimensional plots illustrating the rhIL-7 therapy–induced increased frequency of CD4⁺ RTEs (CD31⁺/CD45RA⁺; red boxes in first row of graphs), and changes in CD4⁺ and CD8⁺ naive, memory, and effector subsets (respectively CD45RA⁺/CD27⁺, CD45RA⁻/CD27⁺, and CD45RA⁻/CD27⁻ sections on second and third rows). (B) Overlay histograms of Ki-67 expression on CD4⁺ RTEs (CD31⁺/CD45RA⁺) and CD4⁺ and CD8⁺ naive (CD45RA⁺/CD27⁺), memory (CD45RA⁻/CD27⁺), and effector (CD45RA⁻/CD27⁻) subsets. Percent Ki-67⁺ cells are indicated in each frame. Red lines, pretreatment; blue lines, day 7; green lines, day 14; orange lines, day 21. Note that at the end of treatment (green line), the percentage of Ki67⁺ memory and effector CD8⁺ populations drop to lower levels than the naive CD8⁺. (C) Two-dimensional plots illustrating the rhIL-7 therapy–induced changes in CD4⁺ (top row) and CD8⁺ (bottom row) naive, central memory, effector memory, and effectors (respectively CD45RA⁺/CCR7⁺, CD45RA⁻/CCR7⁺, CD45RA⁻/CCR7⁻, and CD45RA+/−/CCR7⁻ sections).
… 
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the inability of KGF treatment to increase peripheral regulatory T-
cell numbers. In summary, pre-transplant administration of KGF
can accelerate thymic recovery post allo-HSCT and that the in-
creased export of newly generated T-cells can blunt peripheral ex-
pansion of post-thymic T-cells. However, this thymus-dependent
effect of KGF is insufficient to further ameliorate cGVHD. Never-
theless, the results suggest a potentially important role of KGF in
immune reconstitution and modulation of cGVHD post-allo-
HSCT.
42
IN VIVO EXPANSION OF CD41FOXP31 REGULATORY T CELLS MAY
CONTRIBUTE TO CONTROL OF ACUTE GVHD AFTER HLA-MISMATCHED
ALLOANERGIZED HSCT
Davies, J.
1
, Yuk, D.
1
, Brennan, L.
2
, Nadler, L.
1
, Guinan, E.
2
.
1
Dana
Farber Cancer Institute, Boston, MA;
2
Dana Farber Cancer Institute,
Boston, MA.
Many strategies have been explored to selectively remove allor-
eactive donor T cells to prevent Graft-versus-Host Disease
(GvHD) without impairing immune reconstitution after hemato-
poietic stem cell transplantation (HSCT). An alternative approach
is allostimulation of donor T cells with costimulatory blockade
(CSB) rendering allospecific cells anergized (hyporesponsive to sub-
sequent alloantigenic challenge). Murine and human data suggest
that induction of alloanergy involves cell-mediated suppression, re-
quiring the presence of CD41CD251 regulatory T cells (Tregs).
We conducted a pilot study of haploidentical alloanergized
HSCT after CSB, and measured reconstitution of Treg by intracel-
lular flow cytometry. 5 patients (pts; 4 acute leukemia, one marrow
failure) underwent cyclophosphamide/TBI-conditioned haploi-
dentical HSCT with cyclosporine and methotrexate GvHD pro-
phylaxis. Donor bone marrow was incubated with irradiated
recipient peripheral blood mononuclear cells and anti-B7.1/2 anti-
bodies for 48 hours to induce alloanergy, washed and infused. All pts
engrafted with very rapid reconstitution of T cell subsets, NK cells
and immunoglobulins. All evaluable patients had a marked relative
increase in peripheral blood CD41FOXP31 cells at D 1 20–60.
CD41FOXP31 cells had a memory Treg phenotype (CD251
CD45RO1CTLA41 CD127Lo) and were predominantly HLA
DR- differentiating them from activated T cells. Despite receiving
high doses of donor T cells (median 1.8 (CD4) and 3.1 (CD8)
10
7
/kg) and achieving full donor chimerism, only 2 pts developed
acute GvHD, both Grade II, resolving after short courses of corti-
costeroids. All evaluable patients also had an increase in CD41 T
effector (Teff) cells with an activated phenotype
(CD251HLADR1FOXP3-) at D 1 30–50. Although the antigenic
specificity of Teff was not determined, cytokine secretion may have
led to reversal of anergy and expansion of alloreactive Teff cells.
The marked in vivo expansion of Treg may represent one mecha-
nism of suppressing alloreactive Teff and achieving immunological
control of acute GvHD without impairing immune reconstitution
in pts receiving HLA-mismatched alloanergized donor T cells.
We are using a modification of this strategy in a clinical trial of de-
layed infusion of escalating doses of alloanergized donor T cells af-
ter CD34-selected haploidentical HSCT, to determine the optimal
dose of alloanergized donor T cells that abrogates acute GvHD
without impairment of immune reconstitution.
43
ADMINISTRATION OF rhIL-7 INCREASES TCR REPERTOIRE DIVERSITY
THROUGH PREFERENTIAL EXPANSION OF NAIVE T CELLS
Hakim, F.T.
1
, Memon, S.A.
1
, Sportes, C.
1
, Zhang, H.
2
, Chua, K.
2
,
Fry, T.
2
, Engel, J.
3
, Buffet, R.
3
, Morre, M.
3
, Mackall, C.
2
,
Gress, R.E.
1
.
1
National Cancer Institute, NIH, Bethesda, MD;
2
Na-
tional Cancer Institute, NIH, Bethesda, MD;
3
Cytheris Inc., Rockville,
MD.
Interleukin-7 (IL-7) is a multifunctional cytokine with critical
and non-redundant roles in thymopoiesis and peripheral T-cell ho-
meostasis. We previously reported preliminary results of the first
Phase I study of recombinant human IL-7 (rhIL-7), demonstrating
that two weeks of alternate day treatment with rhIL-7 produced
a marked increase in the number of CD41 and CD81 T cells.
This increase was maintained in follow up assays at 6 to 12 weeks
post treatment. Furthermore, rhIL-7 therapy disproportionately in-
creased CD271CD45RA1 naive cells, which represent the most
diverse elements of the mature T cell receptor (TCR) repertoire,
at the expense of CD27-CD45RA1/- effector populations, which
are often oligoclonal. In CD8 T cells, the proportion of naive cells
increased by 8–39% of total cells. Because of the extent of this pop-
ulation shift, we hypothesized that rhIL-7 treatment would lead to
an overall increase in TCR repertoire diversity in CD41 and
CD81 T-cells. We assessed TCR diversity using spectratype anal-
ysis on sorted CD4 and CD8 populations before and one week after
rhIL-7 therapy (day 21) in six subjects. For each patient, we deter-
mined the divergence of spectratypes in 22 BV families from Gauss-
ian-like normal donor standards and then compared the global
diversity of pre and post spectratypes by Wilcoxon paired non-para-
metric analysis. We determined that rhIL-7 therapy induced a statis-
tically significant increase (P \ .05) in repertoire diversity in either
the CD41, CD81, or both T-cell populations in 4 of the 6 patients.
This enhancement in diversity was particularly remarkable in that
three of these donors were over 60 years of age, and a fourth patient
had reduced lymphocyte populations due to recent chemotherapy.
Given the short duration of therapy, the age of the patients and
the very modest change in TREC we observed, we believe this en-
hancement in diversity was due primarily to differential population
expansion, not IL-7 induced thymic output. Consistent with this in-
terpretation, we observed that a higher percentage of naive T cells
than effector T cells remained in cycle (Ki-671) and maintained el-
evated levels of anti-apoptotic Bcl-2 during IL-7 therapy. We there-
fore propose that rhIL-7 has the potential to induce T-cell growth
and enhance repertoire diversity, even in lympho-depleted patients
with limited thymopoietic capacities, by expanding naive T cell
populations.
44
THE CD4
1
CD25
1
FOXP3
1
COMPARTMENT FOLLOWING CONDITION-
ING AND TRANSPLANT: HOST TREG CELLS EXPAND AND COMPRISE
THE PREDOMINANT COMPONENT FOR SEVERAL MONTHS DURING RE-
CONSTITUTION POST-HCT
Bayer, A.L., Jones, M., Urbieta, M., Chirinos, J., Schreiber, T.,
Armas, L., Thomas, M.R., Levy, R.B. University of Miami Miller School
of Medicine, Miami, FL.
The capacity of CD4
1
CD25
1
Foxp3
1
(Treg) cells to regulate
adaptive and innate immune responses has led to studies investigat-
ing their use in novel strategies to regulate allogeneic T cell re-
sponses during hematopoietic stem cell transplants (HCT). A
fundamental clinical concern post-HCT is the reconstitution of
the lymphoid compartment, particularly T cells which can be excep-
tionally delayed. We have previously found that host Treg cells can
regulate resistance to engraftment following HCT, demonstrating
that such cells survive and function at least transiently in recipients.
The present studies investigated the residual host Treg compart-
ment following varying levels of conditioning (3.0 – 14Gy TBI),
and transplant. We found that recipient CD4
1
CD25
1
Foxp3
1
cells:
1) can survive ablative as well as reduced intensity conditioning, 2)
undergo expansion (BrdU uptake/cell numbers) and 3) contribute
greatly to the Treg compartment for several months post-HCT
during which time donor derived Treg cells gradually arise and
cede this compartment. Within the first 3 weeks post-lethal condi-
tioning and HCT, 95% of the splenic CD4
1
FoxP3
1
cells are pos-
itive for BrdU, vs. 40% in normal mice. Using Thy1.1 congenic
mice, the vast majority of these cells were found to be resistant
(host) Tregs. Two months post-HCT, almost 30% of the compart-
ment was still of host origin. To assess the functional capacity of the
residual Treg cell compartment, we examined development of auto-
immune disease following transplant of IL-2Rb
2/2
BM into synge-
neic recipients. Autoimmune disease was prevented in B6-wt but
not T cell deficient recipients. Interestingly, the failure to transfer
autoimmune disease following IL-2Rb
2/2
HCT into B6-CD4
-/-
recipients was associated with the presence of a peripheral
CD8
1
FoxP3
1
population not detected in B6-wt mice. This finding
18
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[Supplemental Material Index]
Data
July 2008
Claude Sportes · Fran Hakim · Sarfraz Memon · Hua Zhang · Crystal L Mackall
... Moreover, IL7 has been shown to play an important role in the development and preservation of memory immune cell function. Previous work has demonstrated that IL7 can induce naive T-cells to differentiate into a memory phenotype [20,21], and promote the proliferation and activity of effector T-cells against cancer [22][23][24]. In addition, suppressive regulatory T-cells (Tregs) have been shown to express relatively low levels of the IL7 receptor (IL7R) [25]. ...
... IL7 is critical for lymphocyte development and maintenance, and knockout of the IL7R gene leads to a dearth of lymphocyte activity [47]. While IL7 has multiple roles along the T-cell developmental pathway, T-cell survival and maintenance in particular are regulated by IL7 secretion [21,24,48]. Recent research in chimeric antigen receptor (CAR) T-cell engineering has taken advantage of this property to create CAR T-cells that constitutively express IL7. ...
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... While PB CD4 and CD8 T cell counts steadily increased, the proportion of Foxp3 + Treg cells conversely decreased. Additionally, there was a preferential expansion of the TCR repertoire in recent thymic emigrants (RTE) and naïve T cells (41,87). As a key pharmacodynamic characteristic, blood T cell levels blood peaked between day 21 and 28 after CYT99007 administration, while Ki-67 levels in T cells reached its maximum at day 7. ...
... As a key pharmacodynamic characteristic, blood T cell levels blood peaked between day 21 and 28 after CYT99007 administration, while Ki-67 levels in T cells reached its maximum at day 7. Moreover, IL-7 receptor down-regulation continued until day 7, followed by a recovery to basal levels by day 21 and/or day 28 (87). The calculated half-life of unglycosylated IL-7 was between 6 and 10 h (41,88). ...
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Full-text available
  • Feb 2024
The cytokine IL-7 plays critical and nonredundant roles in T cell immunity so that the abundance and availability of IL-7 act as key regulatory mechanisms in T cell immunity. Importantly, IL-7 is not produced by T cells themselves but primarily by non-lymphoid lineage stromal cells and epithelial cells that are limited in their numbers. Thus, T cells depend on cell extrinsic IL-7, and the amount of in vivo IL-7 is considered a major factor in maximizing and maintaining the number of T cells in peripheral tissues. Moreover, IL-7 provides metabolic cues and promotes the survival of both naïve and memory T cells. Thus, IL-7 is also essential for the functional fitness of T cells. In this regard, there has been an extensive effort trying to increase the protein abundance of IL-7 in vivo, with the aim to augment T cell immunity and harness T cell functions in anti-tumor responses. Such approaches started under experimental animal models, but they recently culminated into clinical studies, with striking effects in re-establishing T cell immunity in immunocompromised patients, as well as boosting anti-tumor effects. Depending on the design, glycosylation, and the structure of recombinantly engineered IL-7 proteins and their mimetics, recombinant IL-7 molecules have shown dramatic differences in their stability, efficacy, cellular effects, and overall immune functions. The current review is aimed to summarize the past and present efforts in the field that led to clinical trials, and to highlight the therapeutical significance of IL-7 biology as a master regulator of T cell immunity.
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... Clinically, repeated doses of IL7 have been shown to be well tolerated and can induce T cell expansion with little toxicity (34)(35)(36). CYT107, a glycosylated recombinant IL7 has been widely used and has similar immunological effects (37,38). As IL7 causes the expansion of both CD4 + and CD8 + T cells, this adds an additional benefit to combining IL7 with tLNP in a clinical setting as it could not only augment protein expression, but also increase the total number of T cells, some of which would be transfected with the mRNA cargo. ...
IL7 increases targeted lipid nanoparticle-mediated mRNA expression in T cells in vitro and in vivo by enhancing T cell protein translation
Article
Full-text available
  • Mar 2024
  • P NATL ACAD SCI USA
The use of lipid nanoparticles (LNP) to encapsulate and deliver mRNA has become an important therapeutic advance. In addition to vaccines, LNP-mRNA can be used in many other applications. For example, targeting the LNP with anti-CD5 antibodies (CD5/tLNP) can allow for efficient delivery of mRNA payloads to T cells to express protein. As the percentage of protein expressing T cells induced by an intravenous injection of CD5/tLNP is relatively low (4-20%), our goal was to find ways to increase mRNA-induced translation efficiency. We showed that T cell activation using an anti-CD3 antibody improved protein expression after CD5/tLNP transfection in vitro but not in vivo. T cell health and activation can be increased with cytokines, therefore, using mCherry mRNA as a reporter, we found that culturing either mouse or human T cells with the cytokine IL7 significantly improved protein expression of delivered mRNA in both CD4 ⁺ and CD8 ⁺ T cells in vitro. By pre-treating mice with systemic IL7 followed by tLNP administration, we observed significantly increased mCherry protein expression by T cells in vivo. Transcriptomic analysis of mouse T cells treated with IL7 in vitro revealed enhanced genomic pathways associated with protein translation. Improved translational ability was demonstrated by showing increased levels of protein expression after electroporation with mCherry mRNA in T cells cultured in the presence of IL7, but not with IL2 or IL15. These data show that IL7 selectively increases protein translation in T cells, and this property can be used to improve expression of tLNP-delivered mRNA in vivo.
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... Its signaling in T cells promotes their survival, proliferation, and the formation of memory T cells [66][67][68][69]. IL7 has shown to be less toxic at certain doses compared to other cytokines like IL2, IL12, and IL15 in clinical trials [70][71][72][73]. To deliver IL7 locally, CAR T cells can be designed to secrete it. ...
Chimeric Antigen Receptor T-Cell Therapy for Glioblastoma
Article
Full-text available
  • Nov 2023
Simple Summary Glioblastoma (GBM) is a highly aggressive brain tumor with low survival rates and limited treatment options. This review explores the potential of chimeric antigen receptor (CAR) T-cell immunotherapy as a promising approach for treating GBM. While CAR T-cell therapy has shown success in blood malignancies, it faces challenges when applied to brain tumors, such as tumor heterogeneity and the inhibitory tumor microenvironment. The review discusses existing research and future prospects for CAR T-cell immunotherapy in glioblastoma, highlighting the need for innovative strategies to improve outcomes for patients. Overall, this review provides insights into the current state and future directions of immunotherapy for GBM. Abstract Glioblastoma (GBM), the most common primary brain tumor in adults, is characterized by low survival rates and a grim prognosis. Current treatment modalities, including extensive surgical resection, chemotherapy, and radiation therapy, often yield limited success due to the brain’s sensitivity, leading to significant side effects. Exciting advancements in immunotherapy have recently shown promise in treating various types of tumors, raising hopes for improved outcomes in brain tumor patients. One promising immunotherapy approach is chimeric antigen receptor (CAR) T-cell therapy, which recognizes surface proteins on targeted tumor cells and redirects cytotoxicity towards specific targets. This review aims to discuss the existing research and future prospects for CAR T-cell immunotherapy in treating glioblastoma.
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... 39 In two Phase I dose escalation studies, patients with refractory cancers treated with recombinant human IL-7 (rhIL-7) increased CD4 + and CD8 + T cell counts with increased total numbers of recent thymic emigrants and increased TCR diversity. 40,41 Similarly, increases in T cell counts were observed in patients with metastatic breast cancer treated with rhIL-7. 42 Patients with metastatic castrationresistant prostate cancer tolerated rhIL-7 well and, although not powered to detect clinical efficacy, patients treated with rhIL-7 had elevated antigenspecific humoral and T cell responses. ...
Improving thymus implantation for congenital athymia with interleukin‐7
Article
Full-text available
  • Nov 2023
Objectives Thymus implantation is a recently FDA‐approved therapy for congenital athymia. Patients receiving thymus implantation develop a functional but incomplete T cell compartment. Our objective was to develop a mouse model to study clinical thymus implantation in congenital athymia and to optimise implantation procedures to maximise T cell education and expansion of naïve T cells. Methods Using Foxn1 nu athymic mice as recipients, we tested MHC‐matched and ‐mismatched donor thymi that were implanted as fresh tissue or cultured to remove donor T cells. We first implanted thymus under the kidney capsule and then optimised intramuscular implantation. Using competitive adoptive transfer assays, we investigated whether the failure of newly developed T cells to expand into a complete T cell compartment was because of intrinsic deficits or whether there were deficits in engaging MHC molecules in the periphery. Finally, we tested whether recombinant IL‐7 would promote the expansion of host naïve T cells educated by the implanted thymus. Results We determined that thymus implants in Foxn1 nu athymic mice mimic many aspects of clinical thymus implants in patients with congenital athymia. When we implanted cultured, MHC‐mismatched donor thymus into Foxn1 nu athymic mice, mice developed a limited T cell compartment with notably underdeveloped naïve populations and overrepresented memory‐like T cells. Newly generated T cells were predominantly educated by MHC molecules expressed by the donor thymus, thus potentially undergoing another round of selection once in the peripheral circulation. Using competitive adoptive transfer assays, we compared expansion rates of T cells educated on donor thymus versus T cells educated during typical thymopoiesis in MHC‐matched and ‐mismatched environments. Once in the circulation, regardless of the MHC haplotypes, T cells educated on a donor thymus underwent abnormal expansion with initially more robust proliferation coupled with greater cell death, resembling IL‐7 independent spontaneous expansion. Treating implanted mice with recombinant interleukin (IL‐7) promoted homeostatic expansion that improved T cell development, expanded the T cell receptor repertoire, and normalised the naïve T cell compartment. Conclusion We conclude that implanting cultured thymus into the muscle of Foxn1 nu athymic mice is an appropriate system to study thymus implantation for congenital athymia and immunodeficiencies. T cells are educated by the donor thymus, yet naïve T cells have deficits in expansion. IL‐7 greatly improves T cell development after thymus implantation and may offer a novel strategy to improve outcomes of clinical thymus implantation.
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... Combination approaches have brought clinical success to many and have also improved the overall response rate of patients [101][102][103]. The current approach to cancer treatment through immunotherapy suggests that IL-7 has great potential to treat cancer by increasing the number of circulating CD4+ and CD8+ T cells [104]. ...
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Primary and secondary defects of the thymus
Article
Full-text available
  • Jan 2024
  • IMMUNOL REV
The thymus is the primary site of T‐cell development, enabling generation, and selection of a diverse repertoire of T cells that recognize non‐self, whilst remaining tolerant to self‐ antigens. Severe congenital disorders of thymic development (athymia) can be fatal if left untreated due to infections, and thymic tissue implantation is the only cure. While newborn screening for severe combined immune deficiency has allowed improved detection at birth of congenital athymia, thymic disorders acquired later in life are still underrecognized and assessing the quality of thymic function in such conditions remains a challenge. The thymus is sensitive to injury elicited from a variety of endogenous and exogenous factors, and its self‐renewal capacity decreases with age. Secondary and age‐related forms of thymic dysfunction may lead to an increased risk of infections, malignancy, and autoimmunity. Promising results have been obtained in preclinical models and clinical trials upon administration of soluble factors promoting thymic regeneration, but to date no therapy is approved for clinical use. In this review we provide a background on thymus development, function, and age‐related involution. We discuss disease mechanisms, diagnostic, and therapeutic approaches for primary and secondary thymic defects.
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Persistent inflammation, immunosuppression, and catabolism syndrome (PICS): a review of definitions, potential therapies, and research priorities
Article
Full-text available
  • Jan 2024
  • BRIT J ANAESTH
Persistent Inflammation, Immunosuppression, and Catabolism Syndrome (PICS) is a clinical endotype of chronic critical illness. PICS consists of a self-perpetuating cycle of ongoing organ dysfunction, inflammation, and catabolism resulting in sarcopenia, immunosuppression leading to recurrent infections, metabolic derangements, and changes in bone marrow function. There is heterogeneity regarding the definition of PICS. Currently, there are no licensed treatments specifically for PICS. However, findings can be extrapolated from studies in other conditions with similar features to repurpose drugs, and in animal models. Drugs that can restore immune homeostasis by stimulating lymphocyte production could have potential efficacy. Another treatment could be modifying myeloid-derived suppressor cell (MDSC) activation after day 14 when they are immunosuppressive. Drugs such as interleukin (IL)-1 and IL-6 receptor antagonists might reduce persistent inflammation, although they need to be given at specific time points to avoid adverse effects. Antioxidants could treat the oxidative stress caused by mitochondrial dysfunction in PICS. Possible anti-catabolic agents include testosterone, oxandrolone, IGF-1 (insulin-like growth factor-1), bortezomib, and MURF1 (muscle RING-finger protein-1) inhibitors. Nutritional support strategies that could slow PICS progression include ketogenic feeding and probiotics. The field would benefit from a consensus definition of PICS using biologically based cut-off values. Future research should focus on expanding knowledge on underlying pathophysiological mechanisms of PICS to identify and validate other potential endotypes of chronic critical illness and subsequent treatable traits. There is unlikely to be a universal treatment for PICS, and a multimodal, timely, and personalised therapeutic strategy will be needed to improve outcomes for this growing cohort of patients.
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Increases in CD4 T Lymphocytes with Intermittent Courses of Interleukin-2 in Patients with Human Immunodeficiency Virus Infection — A Preliminary Study
Article
Full-text available
  • Apr 1995
Interleukin-2 is an important regulatory cytokine of the immune system, with potent effects on T cells, B cells, and natural killer cells. In vitro, interleukin-2 can induce the proliferation and differentiation of peripheral-blood mononuclear cells from patients infected with the human immunodeficiency virus (HIV). We treated 25 HIV-infected patients with interleukin-2 administered as a continuous infusion at a dosage of 6 to 18 million IU per day for 5 days every 8 weeks during a period of 7 to 25 months. All patients also received at least one approved antiviral agent. Immunologic and virologic variables were monitored monthly. In 6 of 10 patients with base-line CD4 counts higher than 200 per cubic millimeter, interleukin-2 therapy was associated with at least a 50 percent increase in the number of CD4 cells. Changes ranged from -81 to +2211 cells per cubic millimeter. Interleukin-2 therapy resulted in a decline in the percentage of CD8 lymphocytes expressing HLA-DR and an increase in the percentage of CD4 lymphocytes that were positive for the p55 chain of the interleukin-2 receptor. Four patients had a transient but consistent increase in the plasma HIV RNA level at the end of each infusion. In the remaining 15 patients, who had CD4 counts of 200 or fewer cells per cubic millimeter, interleukin-2 therapy was associated with increased viral activation, few immunologic improvements, and substantial toxic effects. Intermittent courses of interleukin-2 can improve some of the immunologic abnormalities associated with HIV infection in patients with more than 200 CD4 cells per cubic millimeter.
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Lymphopenia in interleukin (IL)-7 gene-deleted mice identifies IL-7 as a nonredundant cytokine
Article
Full-text available
  • May 1995
Interleukin (IL)-7 is a potent stimulus for immature T and B cells and, to a lesser extent, mature T cells. We have inactivated the IL-7 gene in the mouse germline by using gene-targeting techniques to further understand the biology of IL-7. Mutant mice were highly lymphopenic in the peripheral blood and lymphoid organs. Bone marrow B lymphopoiesis was blocked at the transition from pro-B to pre-B cells. Thymic cellularity was reduced 20-fold, but retained normal distribution of CD4 and CD8. Splenic T cellularity was reduced 10-fold. Splenic B cells, also reduced in number, showed an abnormal population of immature B cells in adult animals. The remaining splenic populations of lymphocytes showed normal responsiveness to mitogenic stimuli. These data show that proper T and B cell development is dependent on IL-7. The IL-7-deficient mice are the first example of single cytokine-deficient mice that exhibit severe lymphoid abnormalities.
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Inhibition of murine B and T lymphopoiesis in vivo by an anti- interleukin 7 monoclonal antibody
Article
  • Jul 1993
The effects of interleukin 7 (IL-7) on the growth and differentiation of murine B cell progenitors has been well characterized using in vitro culture methods. We have investigated the role of IL-7 in vivo using a monoclonal antibody that neutralizes IL-7. We find that treatment of mice with this antibody completely inhibits the development of B cell progenitors from the pro-B cell stage forward. We also provide evidence that all peripheral B cells, including those of the B-1 and conventional lineages, are derived from IL-7-dependent precursors. The results are consistent with the rapid turnover of B cell progenitors in the marrow, but a slow turnover of mature B cells in the periphery. In addition to effects on B cell development, anti-IL-7 treatment substantially reduced thymus cellularity, affecting all major thymic subpopulations.
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von Freeden-Jeffry, U., Vieira, P., Lucian, L.A., McNeil, T., Burdach, S.E. & Murray, R. Lymphopenia in interleukin (IL)-7 gene-deleted mice identifies IL-7 as a nonredundant cytokine. J. Exp. Med. 181, 1519-1526
Article
  • Apr 1995
Interleukin (IL)-7 is a potent stimulus for immature T and B cells and, to a lesser extent, mature T cells. We have inactivated the IL-7 gene in the mouse germline by using gene-targeting techniques to further understand the biology of IL-7. Mutant mice were highly lymphopenic in the peripheral blood and lymphoid organs. Bone marrow B lymphopoiesis was blocked at the transition from pro-B to pre-B cells. Thymic cellularity was reduced 20-fold, but retained normal distribution of CD4 and CD8. Splenic T cellularity was reduced 10-fold. Splenic B cells, also reduced in number, showed an abnormal population of immature B cells in adult animals. The remaining splenic populations of lymphocytes showed normal responsiveness to mitogenic stimuli. These data show that proper T and B cell development is dependent on IL-7. The IL-7-deficient mice are the first example of single cytokine-deficient mice that exhibit severe lymphoid abnormalities.
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Loss of IL-7R?? is associated with CD4 T-cell depletion, high interleukin-7 levels and CD28 down-regulation in HIV infected patients
Article
  • Dec 2005
Objective: Elevated levels of interleukin (IL)-7 are present in the blood of HIV-positive patients and it is known that IL-7 receptor (IL-7R)α expression decreases on T cells during HIV infection. The subset(s) of T cells with low IL-7Rα and the consequence of low IL-7Rα expression for T-cell survival are poorly characterized. Design: The frequency of IL-7Rα-negative T cells in HIV-positive patients was studied in relation to CD4 T-cell counts, IL-7 concentration and survival in culture. We analysed IL-7Rα expression in different T-cell populations and in relation to Bcl-2 expression. Methods: Specimens from 38 HIV-1 patients and 17 controls were examined. IL-7Rα and Bcl-2 expression in different T-cell populations was studied by flow cytometry. The influence of IL-7Rα expression on T-cell survival was studied by culturing T cells in the presence of IL-7. Results: Down-regulation of IL-7Rα on T cells correlated with depletion of CD4 T cells (P < 0.001) and also with increased concentration of serum IL-7 (P < 0.05). The decreased IL-7Rα expression was associated with low Bcl-2 expression and with the reduced survival capacity of T cells in the presence of IL-7 in vitro. Particularly, T cells with memory phenotype showed a decreased IL-7Rα expression in association with CD28 down-regulation. Conclusions: The positive effects of IL-7 on survival and homeostatic proliferation of T cells might be severely impaired in HIV-infected individuals due to IL-7Rα down-regulation. Differentiation towards a CD28-negative memory phenotype in response to chronic activation may lead to an overall decrease of IL-7 mediated survival within the peripheral T-cell pool.
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T‐Cell Immunodeficiency Following Cytotoxic Antineoplastic Therapy: A Review
Article
  • Jan 2000
  • STEM CELLS
Although cancer itself is immunosuppressive, cytotoxic antineoplastic therapy is the primary contributor to the clinical immunodeficiency observed in cancer patients. The immunodeficiency induced by cytotoxic antineoplastic therapy is primarily related to T-cell depletion, with CD4 depletion generally more severe than CD8 depletion. Myeloablative therapy, dose-intensive alkylating agents, purine nucleoside analogs, and corticosteroids substantially increase the risk of therapy-induced immunosuppression. Restoration of T-cell populations following cytotoxic antineoplastic therapy is a complex process. Efficient recovery of CD4+ T cell populations requires thymic-dependent pathways which undergo an age-dependent decline resulting in prolonged CD4+ T-cell depletion in adults following T-cell-depleting therapy. Total CD8+ T-cell numbers recover in both children and adults relatively quickly post-therapy; however, CD8+ subset disruptions often remain for a prolonged period. The clinical management of patients with therapy-induced T-cell depletion involves the maintenance of a high index of suspicion for opportunistic pathogens, irradiation of blood products, prophylaxis for viral infections, and reimmunization in selected clinical circumstances. Future research avenues include efforts to rapidly rebuild immunity following cytotoxic antineoplastic therapy so that immune-based therapies may be utilized immediately following cytotoxic therapy to target minimal residual neoplastic disease.
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Interleukin-2 induced immune effects in human immunodeficiency virus-infected patients receiving intermittent interleukin-2 immunotherapy
Article
  • Apr 2001
  • EUR J IMMUNOL
To characterize the immunological effects of intermittent IL-2 therapy, which leads to selective increases in CD4+ T lymphocytes in HIV-infected patients, 11 patients underwent extensive immunological evaluation. While IL-2 induced changes in both CD4+ and CD8+ cell number acutely, only CD4+ cells showed sustained increases following discontinuation of IL-2. Transient increases in expression of the activation markers CD38 and HLA-DR were seen on both CD4+ and CD8+ cells, but CD25 ( chain of the IL-2 receptor) increased exclusively on CD4+ cells. This increase in CD25 expression was sustained for months following discontinuation of IL-2, and was seen in naive as well as memory cells. IL-2 induced cell proliferation, but tachyphylaxis to these proliferative effects developed after 1 week despite continued IL-2 administration. It thus appears that sustained CD25 expression selectively on CD4+ cells is a critical component of the immunological response to IL-2, and that intermittent administration of IL-2 is necessary to overcome the tachyphylaxis to IL-2-induced proliferation.
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IL-7 is a growth and maintenance factor for mature and immature thymocyte subsets
Article
  • Feb 1989
The specific signals inducing the growth and maturation of thymocytes remain undefined. We show here that recombinant IL-7 induces growth of fetal and adult mouse CD4-8- thymocytes. IL-7 also induces a lower but significant response in CD4+8- and CD4-8+ thymocytes. Day 14 fetal thymic lobes cultured in IL-7 for 6 days show a 2-fold increase in cell number when compared to control cultures. The thymocyte subsets that proliferate in response to IL-7 can be maintained in culture for extended periods of time. CD4-8- thymocytes maintained in IL-7 did not change their phenotype with respect to CD4 and CD8 expression. In addition, we show that the combination of IL-7 plus IL-6 provides a potent growth stimulus for CD4+8- and CD4-8+ thymocytes. A cloned thymic epithelial cell, that can be induced to express MHC class II molecules, transcribes both IL-7 and IL-6 mRNA. A cloned thymic macrophage cell line produces IL-6 but no detectable IL-7 mRNA. The pattern of biological activities present in the supernatants of these cell lines is also presented. These observations suggest that the thymic epithelial and macrophage cell types may be an in vivo source of signals which mediate thymocyte development.
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Age, Thymopoiesis, and CD4+ T-Lymphocyte Regeneration after Intensive Chemotherapy
Article
  • Feb 1995
Inadequate reconstitution of CD4+ T lymphocytes is an important clinical problem complicating chemotherapy, human immunodeficiency virus infection, and bone marrow transplantation, but relatively little is known about how CD4+ T lymphocytes regenerate. There are two main possibilities: bone marrow-derived progenitors could reconstitute the lymphocyte population using a thymus-dependent pathway, or thymus-independent pathways could predominate. Previous studies have suggested that the CD45RA glycoprotein on CD4+ T lymphocytes is a marker for progeny generated by a thymus-dependent pathway. We studied 15 patients 1 to 24 years of age who had undergone intensive chemotherapy for cancer. The absolute numbers of CD4+ T lymphocytes in peripheral blood and the expression of CD45 isoforms (CD45RA and CD45RO) on these lymphocytes were studied serially during lymphocyte regeneration after the completion of therapy. Radiographic imaging of the thymus was performed concomitantly. There was an inverse relation between the patients' ages and the CD4+ T-lymphocyte counts six months after therapy was completed (r = -0.92). The CD4+ recovery correlated quantitatively with the appearance of CD45RA+CD4+ T lymphocytes in the blood (r = 0.64). There was a higher proportion of CD45RA+CD4+ T lymphocytes in patients with thymic enlargement after chemotherapy than in patients without such enlargement (two-sided P = 0.015). Thymus-dependent regeneration of CD4+ T lymphocytes occurs primarily in children, whereas even young adults have deficiencies in this pathway. Our results suggest that rapid T-cell regeneration requires residual thymic function in patients receiving high-dose chemotherapy.
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Oligoclonality of tumor-infiltrating lymphocytes from human melanomas
Article
  • Oct 1994
A PCR-based method that determines VDJ junction size patterns in 24 human TCR V beta subfamilies was used to analyze T cells infiltrating sequential malignant melanoma biopsies for the presence of clonal expansions. Infiltrating T cell populations were found to present clonal expansions over a more or less complex polyclonal background. Two clones from a single patient were sequenced and detected in three different tumor sites (skin biopsies), whereas only one of them was also present in peripheral blood. Biopsies from this patient did not show major repertoire changes during in vivo IL-2 treatment. In contrast, in biopsies from a second patient, the expression of all the detected V beta subfamilies was increased and a larger number of clones expanded, probably as a result of therapy. A similar evolution was found among infiltrating T cells cultured in vitro from a third patient for several weeks in the presence of IL-2, where the largely polyclonal repertoire of fresh T cells (from invaded lymph nodes) was dramatically reduced to mainly clonal expansions in all V beta subfamilies detected. The high resolution method used here enables a rapid, comprehensive, qualitative, and semiquantitative description of the T cell repertoire of heterogeneous cell populations. Its use in conjunction with a functional analysis of clones detected within these populations should provide a better understanding of the evolution of the T cell repertoire among tumor-infiltrating lymphocytes during the progression of the disease and as a response to immunotherapy.
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