Download Bulletin 933B: Hematopoietic Stem Cell Transplantation

Bulletin 9338
Hematopoietic Stem Cell Transplantation
Reconstitution of T Cell Immune Function
Pat Roth, Ph.D., Ronald Paul, Ph.D.
Enrique Rabellino, M.D.
INTRODUCTION
T CELL BIOLOGY
Transplantation of hematopoietic stem cells (HSC) has
been applied successfully to restore bone marrow function
and provide curative therapy for a number of previously
untreatable diseases. The original HSC transplant
programs were aimed at reconstituting
hemato-lymphopoiesis in patients with immunodeficiency
syndromes, aplastic anemia and acute leukemia.1
Encouraged by advances in myeloablative chemotherapy
and immunosuppressive regimens, HSC transplant
protocols were subsequently developed for many other
hematological malignancies, solid tumors (breast, ovarian,
neuroblastomas etc.) and more recently, for a host of
metabolic, congenital and autoimmune disorders.2-4
Helper CD4 and suppressor CD8 lymphocytes were the
first human T cell subsets characterized by
immunophenotyping. These findings gained clinical
relevance with the recognition of the effect of HIV infection
on T cells and the importance of CD4 monitoring in the
management of HIV/AIDS patients. CD4 phenotyping
provides critical clinical information concerning diagnosis,
prognosis, monitoring and evaluating the efficacy of
developing therapies.
HSC transplantation has the capacity to reconstitute
erythroid, myeloid and lymphoid functions.The pluripotent
nature of HSC give rise to all blood cell types including
immune cells. Patient pre-conditioning for stem cell
transplantation includes a myeloablation that eliminates
most of the lymphoid cells as well as the lymphopoietic
tissues. Thus, the reconstitution of the immune system and
in particular the recovery of T cell function, is one of the
most important goals of stem cell transplantation.
T cells are regarded as the key regulatory and effector
cells for multiple cellular and humoral immune functions.
The availability of monoclonal antibodies to lymphoid
antigens has played a key role in the identification and
characterization of functional T cell populations. Therefore
the dissection of T cell responses has led to our current
understanding of the functional heterogeneity among
T cells and their associated phenotypes. Thus, the
enumeration of T cells has evolved from T cell counting to
subset profiling.
The purpose of this communication is to review the current
understanding of reconstitution of immunocompetent cells
and the methods used to assess their recovery.
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Japan, Tokyo (81) 3 5404 8424. Latin America (1) (305) 380 4709. Mexico, Mexico City (525) 575 6805. Netherlands, Mijdrecht (31) 297 230630. Singapore (65) 6339 3633.
South Africa/Sub-Saharan Africa, Johannesburg (27) 11 805 2014. Sweden, Bromma (46) 8 564 85 900. Switzerland, Nyon 0800 850 810. Taiwan, Taipei (886) 2 2378 3456.
Turkey, Istanbul (90) 216 309 1900. UK, High Wycombe (44) 01494 441181. USA, Miami, FL (1) 800 526 3821, Option 7.
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The significance of these findings in conjunction with the
availability of monoclonal reagents and technological
advances in flow cytometry led to an explosion in the
research of T lymphocyte differentiation and functional
development. This research has led to the current
understanding of T cell ontogeny including the recognition
of naïve, memory and effector T cells. Due to this new
understanding of T cell heterogeneity, the laboratory
evaluation of T cells must be more comprehensive than the
simple enumeration of CD4 and CD8 subsets.
IMMUNE RECONSTITUTION
Immune reconstitution following stem cell transplantation
encompasses the recovery of the adaptive cells and
functions of the immune system. Sustained and complete
immune reconstitution is achieved in stages, many of
which have been defined by the appearance of distinct cell
populations.
LYMPHOCYTE SUBSETS
In order to evaluate T cell recovery, the first level of posttransplant monitoring consists of immunophenotyping the
total T cell population and the helper (CD4) and suppressor
(CD8) subsets (Figure 1).
Hematopoietic Stem Cell Transplantation
Reconstitution of T Cell
Immune Function
Figure 1
A study of the kinetics of CD4+ T cell recovery
demonstrated that the first wave of cells is exclusively in
the CD4+CD45RA-CD45R0+ memory subset. These
memory cells resulted from the expansion of donor-derived
naïve/memory T cells present in the inoculum. However,
repopulation of naïve CD4 T cells (CD4+CD45RA+CD45R0-)
followed different kinetics. Naïve cells develop from T
lineage committed stem cells which undergo differentiation
in the thymus and which repopulate a broad T cell
repertoire. The effect of age on the naïve cell repopulation
is consistent with the decrease in thymic function with
age.15 The fact that early expansion of CD4 cells was not
dependent on patient age supports the notion that this
expansion included mostly memory cells. This explains why
young recipients recovered normal levels of naïve cells
after the first year while for many older patients with low
thymic function, it required years to detect any naïve cells.
900
CD4
Absolute cell number/ul
800
CD8
700
B
600
NK
500
400
300
200
100
0
0
3
6
9
12
18
24
Months after transplantation
Figure 1 Recovery of lymphocyte subsets over a two year
post transplant period. This data, compiled from various studies,
demonstrates the recovery profiles of T cell subsets. B and NK cell
recovery which are also included for comparison are beyond the
scope of this discussion.
In the T cell compartment, CD8+ cells are reconstituted
earlier than CD4+ cells. For a variety of transplant
conditions, CD8+ T cells approach normal levels within the
first 6 months after stem cell infusion.5-11 However, in each
of these studies, CD4+T cell levels in adults remained
abnormally low up to a year after transplant. One study
demonstrated low CD4 levels at the 5-year follow-up.7
CD8+ T cell counts in children rebounded somewhat faster
than for adults, and CD4+T cells were up to normal levels
by the end of the first year after transplant.6;9;12
Several studies demonstrated greater efficiency in T
lymphocyte recovery when donor HSC were derived from
peripheral blood as compared to bone marrow.13
Presumably, this was due to the higher content of
committed T cells available in the transplanted peripheral
blood (PB) sample. In addition, transplantation of
peripheral blood stem cells significantly lowered the
incidence of infection13, suggesting some association
between this parameter and efficient T cell recovery.10
These data indicate that this first level of phenotypic
screening has provided useful preliminary information on T
lymphocyte recovery following HSC transplantation.
ANALYSIS OF FUNCTIONAL
LYMPHOCYTE SUBSETS
While quantitation of the major lymphocyte subsets
indicates the initial commitment of stem cells to the
lymphoid lineage, enumeration of these broad cell
populations does not always correlate with the incidence of
post transplant complications. The next level of analysis
further dissects these major populations into subsets that
are associated with specific immune functions.
Following migration of lymphoid-committed stem cells,
T cells maturation occurs in the thymus (Figure 2).
Mature T cells are released from the thymus as naïve
CD4 or CD8 cells. Acquisition of effector and memory
functions occur upon antigen stimulation. Effector
T cells are generally short -lived. The memory population
comprises a pool of long-lived immune cells that are
primed for specific responses.
Figure 2
Other studies showed below normal levels of naïve cells in
adults 1-5 years after transplantation.7;6 Age dependence
of naïve cell recovery has been shown for different
transplant variables, such as source of stem cells,9;12;16 and
incidence of Graft Versus Host Disease (GVHD).16 It is
important to note that these variables are all inter-related
and it is often difficult to determine which variable is
independent. Overall, the integrity of the thymic
environment is likely to be most important factor
associated with the age-dependence of naïve
T cell reconstitution.
Monitoring the recovery of naïve CD4+ T cell levels has
become one of the standard methods for evaluating the
efficacy of transplant protocols. Various studies have
shown that the levels of naïve CD4 cells are higher when
either cord blood or mobilized peripheral blood is the
source of transplanted HSC as compared to bone
marrow.10, 13, 14, 17 It is also believed that transplantation
of relatively immature and naïve cord blood stem cells
reduces the incidence of GVHD, thus the use of cord blood
(CB) stem cells is being carefully evaluated. Naïve
cell monitoring is a large component of this evaluation.
The expression of CD45RA and CD45R0 was initially used
as the basic phenotypic profile to distinguish naïve and
memory T cell subsets, respectively. More recently,
CD45RA and CD45R0 were found to overlap in different
functional subsets.17; 18 Thus the combination of CD8 or
CD4 and CD45RA with a variety of other markers is
necessary to further discriminate functional T cells.
Table 1 describes both the basic and expanded profiles
that have been used to define naïve, memory and effector
T cell populations. As an example, expression of CD27 on
CD8 CD45RA+ cells correlated with a functionally naïve
population and loss of CD27 was seen when these cells
became highly differentiated19. Figure 3 illustrates an
example of this expanded phenotypic analysis. Similar
results were found with other marker combinations.19-20;21
22Thus, phenotypic analysis of naïve/memory cells should
include CD45RA as well as other markers that refine the
definition of these functional subsets.23
Table 1
Functional T Cell
Subset
Basic Phenotypic
Profile
Expanded Phenotype
Profile
Naïve CD4 T cells
CD4+CD45RA+
or
CD4+CD45R0-
CD11a low
CD27+
CD62L hi
CCR7+
Memory CD4 T cells
CD4+CD45RAor
CD4+CD45R0+
CD27CD62L
CCR7-
Effector CD4 T cells
CD4+CD45RA+
or
CD4+CD45R0+
Cytokines (post-stimulation)
Naïve CD8 T cells
CD8+CD45RA+
or
CD8+CD45R0-
CD11a low
CD31+ (TREC hi)
CD27+
CD62L hi
CCR7+
CD244/2B4-
Memory CD8 T cells
CD8+CD45RAor
CD8+CD45R0+
CD27CD62L
CCR7-
Effector CD8 T cells
CD8+CD45RA+
or
CD8+CD45R0+
CD27CD62L low
CCR7CD244/2B4+
Perforin, Granzyme B +
Cytokines (post-stimulation)
Activated T cells
CD4+CD25+, CD69+,
CD71,HLA-DR+
Effector cells (T helper and T cytotoxic)
Pluripotent (HSC) stem cells →T lineage stem cells → Thymus → Naïve T cells (CD4 and CD8)
Memory (CD4 and CD8)
The reconstitution of functional T cell subsets has been
extensively examined. Studies suggest these cells affect
the incidence of post-transplant infection.10;14 The focus of
many studies has been on naïve and memory T cell
subsets, since the reappearance of a broad naïve T cell
repertoire is thought to be necessary to respond to a
lifetime of antigenic stimulation.
CD8+CD25+, CD69+,
CD71, HLA-DR+, CD38+
3
low
low
Hematopoietic Stem Cell Transplantation
Reconstitution of T Cell
Immune Function
Figure 3
T CELL RECEPTOR DIVERSITY
Another parameter that is increasingly useful in monitoring
T cell reconstitution is T cell receptor (TCR) diversity. The
ability of the immune system to protect an individual from
a great variety of pathogens is dependent on a diverse
TCR repertoire. Diversity is generated during T cell
maturation in the thymus, by recombining TCR genes in
the variable (V) regions.
CDR3 Spectratyping
Further
phenotypic
analysis
CDR3 spectratyping analyzes the complexity of the
complementarity-determining regions (CDR3) of the TCR
Vβ. These regions are generated during gene
rearrangement in the thymus and play a major role in
conferring TCR diversity. The greater the size
heterogeneity of CDR3 sequences demonstrated for each
Vβ family, the more complex and diverse is the TCR
repertoire. During the course of T cell reconstitution the
number of peaks in a CDR3 spectragraph would be
expected to increase as has been described in Roux et
al.15 This study also demonstrated a strong correlation
between the number of CDR3-complete Vβ families and the
number of CD4+CD45RA+ CD45R0- T cells. Of clinical
importance, an increase in naïve cells and in their diversity
was necessary for patients to respond to tetanus toxoid
immunization. This finding may help define more
effective revaccination protocols. These data also
suggests that the degree of diversity is indicative of the
level of immune protection.
T Cell Receptor Excision Circles (TREC)
Figure 3 Functional subpopulations of T cell subsets.
The definitions of naïve, memory and effector T cell populations
are based upon the expression of CD45RA and CD27.
During thymic TCR gene rearrangement, DNA fragments
are excised in the form of circular DNA or TRECs. TRECs
are stable within the cell but do not replicate and are
diluted during T cell proliferation. Quantitative detection of
TRECs using PCR appears to be a sensitive measurement
of thymic T cell output. T cells have the largest number of
circles right after rearrangement, then get diluted out by
cell division as cells get further in time from thymic
emigration. This measurement has been used to monitor
the emergence of naïve T cells after stem cell
transplantation. TREC levels have been shown to correlate
with the frequency of naïve CD4 T cells and with the
recovery of proliferative responses.12
5
Hematopoietic Stem Cell Transplantation
Reconstitution of T Cell
Immune Function
Figure 4
Tube A
L
Vβ5.3
Vβ9
K
K
Vβ17
Vβ7.1
M
Vβ3
M
Tube C
L
NonActivated
CD4
Vβ13.1
Vβ5.1
K
SS
Vβ13.6
Vβ20
M
Vβ8
PE
M
Vβ16
Tube D
Vβ18
L
K
Cytometric analysis has previously limited antibody based
T cell repertoire monitoring, because of the large number
of cells needed to test more than 20 Vβ families. However
a recent advance, which combined most of the available
Vβ antibodies into 8 tubes24 has greatly improved this
methodology and made it feasible to include it in the
routine monitoring of immune reconstitution.
Tube B
CD69
L
CD3
It is now possible to measure the diversity of the T cell
repertoire by detection of the distinct Vβ families of the
TCR β chain using multiparametric flow cytometric
analysis. This approach can determine whether
representation in the TCR repertoire is broad or
skewed/biased. The broadest Vβ usage is indicative of the
most intact immune system. The imbalance in
naïve/memory T cells seen following stem cell
transplantation undoubtedly contributes to a bias in
Vβ usage.
Figure 5
CD3
Phenotypic Analysis Of The
Vβ Repertoire
IFN ϒ
CD8
Tube E
Tube F
L
L
Vβ5.2
Vβ23
K
K
Vβ2
Vβ1
M
Vβ12
Vβ21.3
Tube H
Vβ11
Flow cytometry offers a standardized method for the
evaluation of TCR Vβ diversity that is easier, less
expensive and faster than comparable molecular
methods. In addition, multiparametric analysis permits the
evaluation of TCR Vβ diversity within functional T cell
subsets. This capability may contribute to better patient
management in the evaluation of HSC transplantation and
immune reconstitution.
IMMUNE FUNCTION/RESPONSE
The measurement of T cell responses after in vitro
stimulation is often used to assess effector functions
during immune reconstitution. Measurement of proliferative
responses has traditionally been performed by measuring
thymidine incorporation following polyclonal stimulation
with mitogens (PHA, PWM or anti-CD3)7;8;9;12 or specific
anti-viral and bacterial antigens.7;9;12;15 In general, it has
been shown that proliferative responses correlate with the
generation of naïve T cells and a high degree of diversity
as found using phenotyping or molecular methods. More
recently, single cell analysis of proliferation using reagents
such as BrdU, PCNA, Ki67 and CFSE has become popular
in an effort to eliminate radioisotope-based protocols.25
L
K
Vβ13.2
Activated
K
Vβ4
Vβ22
M
Vβ14
M
CD69
Tube G
L
CD3
Figure 4 illustrates the use of Betamark™ flow cytometry
to quantitate the expression of 24 distinct TCR Vβ families
in functional T cell populations.
CD3
M
CD4
Vβ7.2
FITC
Figure 4 Analysis of TCR Vβ diversity in functional T cell subsets.
Twenty four Vβ families were characterized within the naïve CD4+ T cell
population as defined by expression of CD45RA and CD27. See Figure 2
This analytical approach provides a comprehensive assessment of T cell
diversity within the CD4 and CD8 subsets.
SS
CD8
IFN ϒ
Figure 5 Analysis of Intracellular IFNγ in Peripheral Blood
Mononuclear Cells (PBMC). PBMC were analyzed for CD69 and
IFNγ co-expression following stimulation with Staphylococcal
enterotoxin B (SEB) and anti-CD28. Sample preparation and staining
was identical for the non-activated control. Samples were
permeabilized with IntraPrep and stained with a 5-color antibody
cocktail consisting of CD8/IFNγ/CD4/CD69/CD3.
7
Hematopoietic Stem Cell Transplantation
Reconstitution of T Cell
Immune Function
Multiparametric flow cytometry is the method of choice in
assessing the activation responses of lymphocytes, as
many post stimulation events consist of up regulation of
surface antigens and intracellular antigens that can be
detected with monoclonal antibodies.25 Thus, measurement
of CD69 and cytokine production hours after stimulation,
or CD25, CD71, HLA-DR, etc. days after stimulation
provides an activation profile which may reflect T cell
functional status during immune reconstitution. An example
of intracellular cytokine analysis is shown in Figure 5.
SUMMARY
Transplantation of Hematopoietic stem cells (HSC) has
become a common procedure for the treatment of a
number of hematological, neoplastic, metabolic and
genetic disorders
Following transplantation, reconstitution of immune function
is achieved in stages as revealed by the enumeration of
distinct lymphocyte populations. Immunophenotyping
studies have shown that CD8+ lymphocytes are recovered
faster than CD4+ cells. Acquisition of different
immunological functions is slow and depends on many
variables including source of stem cells, HSC quantity,
quality of the inoculum, clinical conditions, patient age, and
graft complications (GVHD, infections).
Immunophenotyping by multiparametric flow cytometric
analysis has been very effective in defining functional
subsets of lymphocytes and has allowed the phenotypic
characterization of naïve/memory and effector cells. In
addition, T cell diversity can be assessed by TCR Vβ
analysis using flow cytometry. This capability has led to
more comprehensive immunological monitoring of grafted
patients. As a result, it is now apparent that one of the
most critical parameters is the re-appearance of newly
generated and fully diverse naïve T cells, reflecting the
availability of T lineage stem cells and the level of thymic
function. Once an intact T cell compartment is
phenotypically identified, functional integrity of the immune
cells can be confirmed by measuring post-stimulation
parameters which are indicative of responsiveness.
The improved ability to monitor the course of immune
reconstitution will aid in devising therapeutic strategies for
HSC transplantion and post-transplant management.
References
(1) Cao,T.M., C.Kusnierz-Glaz, F.Valone, K.E.Stockerl-Goldstein, W.W.Hu,
L.Johnston, K.G.Blume, S.Strober, and R.S.Negrin. 2001. Rapid
engraftment after allogeneic transplantation of density-enriched
peripheral blood CD34+ cells in patients with advanced hematologic
malignancies. Cancer 91:2205-2213.
(2) Atkinson K. 2000. Clinical Bone Marrow and Blood Stem Cell
Transplantation. Cambridge University Press, Boston.
(3) Little,M.T. and R.Storb. 2002. History of haematopoietic stem-cell
transplantation. Nature Reviews Cancer 2:231-238.
(4) Burt,R.K., A.Fassas, J.Snowden, J.M.van Laar, T.Kozak,
N.M.Wulffraat, R.A.Nash, C.E.Dunbar, R.Arnold, G.Prentice,
S.Bingham, A.M.Marmont, and P.A.McSweeney. 2001. Collection of
hematopoietic stem cells from patients with autoimmune diseases.
Bone Marrow Transplant. 28:1-12.
(5) Douek,D.C., R.A.Vescio, M.R.Betts, J.M.Brenchley, B.J.Hill, L.Zhang,
J.R.Berenson, R.H.Collins, and R.A.Koup. 2000. Assessment of thymic
output in adults after haematopoietic stem-cell transplantation and
prediction of T-cell reconstitution. Lancet 355:1875-1881.
(6) Savage,W.J., J.J.Bleesing, D.Douek, M.R.Brown, G.M.Linton,
H.L.Malech, and M.E.Horwitz. 2001. Lymphocyte reconstitution
following non-myeloablative hematopoietic stem cell transplantation
follows two patterns depending on age and donor/recipient
chimerism. Bone Marrow Transplant. 28:463-471.
(7) Fujimaki,K., A.Maruta, M.Yoshida, F.Kodama, M.Matsuzaki,
S.Fujisawa, H.Kanamori, and Y.Ishigatsubo. 2001. Immune
reconstitution assessed during five years after allogeneic bone
marrow transplantation. Bone Marrow Transplant. 27:1275-1281.
(8) Morecki,S., Y.Gelfand, A.Nagler, R.Or, E.Naparstek, G.Varadi,
D.Engelhard, A.Akerstein, and S.Slavin. 2001. Immune reconstitution
following allogeneic stem cell transplantation in recipients
conditioned by low intensity vs myeloablative regimen. Bone Marrow
Transplant. 28:243-249.
(9) Small,T.N., E.B.Papadopoulos, F.Boulad, P.Black, H.Castro-Malaspina,
B.H.Childs, N.Collins, A.Gillio, D.George, A.Jakubowski, G.Heller,
M.Fazzari, N.Kernan, S.MacKinnon, P.Szabolcs, J.W.Young, and
R.J.O'Reilly. 1999. Comparison of Immune Reconstitution After
Unrelated and Related T-Cell-Depleted Bone Marrow Transplantation:
Effect of Patient Age and Donor Leukocyte Infusions. Blood 93:467480.
(10) Storek,J., M.A.Dawson, B.Storer, T.Stevens-Ayers, D.G.Maloney,
K.A.Marr, R.P.Witherspoon, W.Bensinger, M.E.Flowers, P.Martin,
R.Storb, F.R.Appelbaum, and M.Boeckh. 2001. Immune
reconstitution after allogeneic marrow transplantation compared
with blood stem cell transplantation. Blood 97:3380-3389.
(11) Wu,C.J., A.Chillemi, E.P.Alyea, E.Orsini, D.Neuberg, R.J.Soiffer,
and J.Ritz. 2000. Reconstitution of T-cell receptor repertoire
diversity following T-cell depleted allogeneic bone marrow
transplantation is related to hematopoietic chimerism. Blood
95:352-359.
(12) Klein,A.K., D.D.Patel, M.E.Gooding, G.D.Sempowski, B.J.Chen,
C.Liu, J.Kurtzberg, B.F.Haynes, and N.J.Chao. 2001. T-Cell recovery
in adults and children following umbilical cord blood
transplantation. Biol.Blood Marrow Transplant. 7:454-466.
(13) Ottinger,H.D., D.W.Beelen, B.Scheulen, U.W.Schaefer, and H.GrosseWilde. 1996. Improved immune reconstitution after
allotransplantation of peripheral blood stem cells instead of bone
marrow. Blood 88:2775-2779.
(14) Elmaagacli,A.H., S.Basoglu, R.Peceny, R.Trenschel, H.Ottinger,
A.Lollert, V.Runde, H.Grosse-Wilde, D.W.Beelen, and U.W.Schaefer.
2002. Improved disease-free-survival after transplantation of
peripheral blood stem cells as compared with bone marrow from
HLA-identical unrelated donors in patients with first chronic phase
chronic myeloid leukemia. Blood 99:1130-1135.
(15) Roux,E., F.Dumont-Girard, M.Starobinski, C.A.Siegrist, C.Helg,
B.Chapuis, and E.Roosnek. 2000. Recovery of immune reactivity
after T-cell-depleted bone marrow transplantation depends on
thymic activity. Blood 96:2299-2303.
(16) Dumont-Girard,F., E.Roux, R.A.van Lier, G.Hale, C.Helg, B.Chapuis,
M.Starobinski, and E.Roosnek. 1998. Reconstitution of the T-Cell
Compartment After Bone Marrow Transplantation: Restoration of
the Repertoire by Thymic Emigrants. Blood 92:4464-4471.
(17) Talvensaari,K., E.Clave, C.Douay, C.Rabian, L.Garderet, M.Busson,
F.Garnier, D.Douek, E.Gluckman, D.Charron, and A.Toubert. 2002. A
broad T-cell repertoire diversity and an efficient thymic function
indicate a favorable long-term immune reconstitution after cord
blood stem cell transplantation. Blood 99:1458-1464.
(18) Faint,J.M., N.E.Annels, S.J.Curnow, P.Shields, D.Pilling, A.D.Hislop,
L.Wu, A.N.Akbar, C.D.Buckley, P.A.Moss, D.H.Adams, A.B.Rickinson,
and M.Salmon. 2001. Memory T cells constitute a subset of the
human CD8+CD45RA+ pool with distinct phenotypic and migratory
characteristics. J Immunol 167:212-220.
(19) Baars,P.A., L.M.Ribeiro Do Couto, J.H.Leusen, B.Hooibrink,
T.W.Kuijpers, S.M.Lens, and R.A.van Lier. 2000. Cytolytic
mechanisms and expression of activation-regulating receptors on
effector-type CD8+CD45RA+. J Immunol 165:1910-1917.
(20) Champagne,P., G.S.Ogg, A.S.King, C.Knabenhans, K.Ellefsen,
M.Nobile, V.Appay, G.P.Rizzardi, S.Fleury, M.Lipp, R.Forster,
S.Rowland-Jones, R.P.Sekaly, A.J.McMichael, and G.Pantaleo. 2001.
Skewed maturation of memory HIV-specific CD8 T lymphocytes.
Nature 410:106-111.
(21) Chen,G., P.Shankar, C.Lange, H.Valdez, P.R.Skolnik, L.Wu,
N.Manjunath, and J.Lieberman. 2001. CD8 T cells specific for
human immunodeficiency virus, Epstein-Barr virus, and
cytomegalovirus lack molecules for homing to lymphoid sites of
infection. Blood 98:156-164.
(22) Kimmig,S., G.K.Przybylski, C.A.Schmidt, K.Laurisch, B.Mowes,
A.Radbruch, and A.Thiel. 2002. Two subsets of naive T helper cells
with distinct T cell receptor excision circle content in human adult
peripheral blood. J.Exp.Med. 195:789-794.
(23) Speiser,D.E., M.Colonna, M.Ayyoub, M.Cella, M.J.Pittet, P.Batard,
D.Valmori, P.Guillaume, D.Lienard, J.C.Cerottini, and P.Romero.
2001. The activatory receptor 2B4 is expressed in vivo by human
CD8+ effector alpha beta T cells. J Immunol 167:6165-6170.
(24) De Rosa,S.C., L.A.Herzenberg, L.A.Herzenberg, and M.Roederer.
2001. 11-color, 13-parameter flow cytometry: identification of
human naive T cells by phenotype, function, and T-cell receptor
diversity. Nat.Med. 7:245-248.
(25) van den,B.R., P.P.Boor, E.G.van Lochem, W.C.Hop, A.W.Langerak,
I.L.Wolvers-Tettero, H.Hooijkaas, and J.J.van Dongen. 2000. Flow
cytometric analysis of the Vbeta repertoire in healthy controls.
Cytometry 40:336-345.
(26) Darzynkiewicz,Z.C.H.a.&.R.J.P. 2001. Cytometry - Methods in Cell
Biology. Academic Press, San Diego, CA.
9
Hematopoietic Stem Cell Transplantation
Reconstitution of T Cell
Immune Function
PRODUCT LISTING FOR IMMUNE RECONSTITUTION STUDIES
MULTI-COLOR PRODUCTS FOR FLOW CYTOMETRY
SINGLE COLOR PRODUCTS FOR FLOW CYTOMETRY
Specificity
FREEZE DRIED
Application
CD3
LS
CD4
CD8
CD11a
LS
LS
NME
COULTER CLONE/IOTest
Clone
Purified
UCHT1
6604629
Biotin
A
BL4
13B8.2
IM0115
IM0263
IM0398
IM0704
SFCI12T4D11
6602138 6602393
SFCI21Thy2D3
6602139
B9.11
IM0102
25.3
IM0157
B1.49.9
IM0119
33b3.1
IM0317
1A4CD27
IM2034
CD28
NME
CD28.2
IM1376
NME
1F11
IM2052
IM0447
T16
A
6602385
NME
CD45R0
NME
CD62L
NME
CD69
CD71
IM0266
IM2635
APC
PC7
IM2467 6607100
FITC
RD1
ECD
6604623 6604621
IM0448
IM0451
IM0452
IM0860
IM1433
IM0477
IM0478
IM1235
IM1236
37L (T9)
6603117
A
C1.7
IM1607
2B1
IM1517
B8.12.2
IM0108
A
Immu-357
IL-2
EF
N7.48A
IL-4
EF
4D9
Ki-67
P
Ki67
INF-Gamma
P
45.15
PCNA
P
5A10
TNF-Alpha
EF
188
IM2646
6607107
IM2071 6607111 6607108
IM1832
IM2651
6604928
IM0584
IM1834
IM1247
IM1307
IM1230
IM1231
6603904 6603866
IM2712
IM2214
IM2713
Part Number
FITC
PE/RD1
ECD
PC5
LS
6607013
X
X
X
X
PC5
CD45/56/19/3
LS
6607073
X
X
X
X
6607010
CD8/4/3
LS
6607053
X
X
X
CD45//4/3
LS
6607015
X
X
X
CD45/8/3
LS
6607017
X
X
X
CD8/4/3
LS
6607103
X
X
X
CD62L/45RA/4
NME
6607104
X
X
X
CD62L/45RA/8
NME
6607105
X
X
X
CD62L/45R0/8
NME
6607106
X
X
CD8/4/3
LS
IM3485
X
X
X
CD16/56/3
LS
IM3487
X
X
X
CD25/4/3
LS
IM3486
X
X
X
CD3/4/45v
LS
IM1651
X
X
X
CD3/8/45
LS
IM1670
X
X
X
CD4/8/3
LS
IM1650
X
X
X
CD4/8/45
LS
IM1653
X
X
X
CD3/4
LS
6604929/IM1382/IM1287
X
X
CD3/8
LS
6604930/IM1310/IM1383
X
X
CD4/8
LS
6604614/IM0747/IM1385
X
X
CD4/CD45RA
NME
6603800
X
X
CD8/CD4
LS
6603802/IM1379
X
X
IM1943 6607110
IM0482
IM0483
IM0462
IM0463
IM1638
6602924
IM2667
X
X
A
IM1295/IM2053/IM1390
X
X
CD8/38
A
IM2695
X
X
CD38/8
A
IM2673
X
X
IM2656
HLA-DR/3
A
IM1300
X
X
HLA-DR/8
A
IM1199
X
X
IM2658
IM0464
IM1639
IM3636
IM2659
MULTI-COLOR PRODUCTS FOR FLOW CYTOMETRY
IM3635
6602920
IM0109
6603140 6603424
A
6603450
Specificities
Application
Part Number
IOTest Beta MarkTCR Vβ Repertoire Kit
D
M3497
6604366
IM2718
IM1316
IM2719
MISCELLANEOUS PRODUCTS
IM2716
IM2717
IM1510
IntraPrep Permeabilization Reagent
IM2388/IM2389
Flow-Count Fluorospheres
7547053
IM3279
Activation
Naive, Memory Effector
Proliferation
LS
Lymphocyte Subsets
EF
Effector Function
D
Diversity
11
PC7
X
IM2001
IM1608
IM0261
CD3/25
CD3/HLA-DR
IM2655
6604077
P
BL2
IM0479 6607112
IM1246
IM0780
BrdU
9-49 (I3)
IM2469
6602944
IM2117
A
6603862 6603850 6604727
6607102
6603181 IM2711
IM0537
YDJ1.2.2
“HLA-DR,DP,DQ”
IM2638
IM2371
DREG56
A
6607101
IM1431
IM0775
SFCI28T17G6 (TQ1)
TP1.55.3
IM2468
IM2409
UCHL1
A
IM2636
IM2578
LS198-4-3
ALB11
IM0449
IM0481
H279 (I2)
P
PC5
IM2705
6604422
IM0366
CD244
HLA-DR
ECD
IM1282
6603861 6603848 6604728 6607011
IM0450
2H4LDH11LDB9 (2H4)
CD45RA
PE/RD1
6602864
5.6E
CD38
FITC
6603200
NME
CD31
Purified
IM0178
CD27
A
Application
CD45/4/8/3
CYTOSTAT
6604701
1HT44H3(IL2R1)
CD25
FITC
IM1301 6604625 6604629 IM1281
HIT3a
X35
NME
Specificities
LIQUID